Folded recombinant catalytic fragments of multidomain serine proteases, preparation and uses thereof

ABSTRACT

The invention relates to unglycosylated folded C-terminal fragments of a multidomain serine protease of the complement cascade obtainable by expression in a bacterial host, wherein said serine protease is capable of binding a recognition molecule of the complement cascade, e.g. C1 or MBL. The invention also relates to methods and bacterial expression vectors for the preparation of said fragments, uses of said fragments for raising antibodies and screening substrates or inhibitors of said serine proteases and uses of the fragments in research and treatment of complement related disorders. The invention also relates to assay methods for assessing MASP-1 and MASP-2 levels in a sample of biological origin. 
     The invention provides for research tools, assays and diagnostic kits useful in complement research and research and diagnosis of complement related disorders.

This is a divisional of application Ser. No. 10/636,602, filed Aug. 8,2003, and claims priority to provisional application Ser. No.60/401,755, filed Aug. 8, 2002. The entire content of the twoaforementioned applications is hereby incorporated by reference herein.

FIELD OF THE INVENTION

The invention relates to unglycosylated folded C-terminal fragments of amultidomain serine protease of the complement cascade obtainable byexpression in a bacterial host, wherein said serine protease is capableof binding a recognition molecule of the complement cascade, e.g. C1 orMBL. The invention also relates to methods and bacterial expressionvectors for the preparation of said fragments, uses of said fragmentsfor raising antibodies and screening substrates or inhibitors of saidserine proteases and uses of the fragments in research and treatment ofcomplement related disorders. The invention also relates to assaymethods for assessing levels of multidomain complement serine proteasesin samples of biological origin.

BACKGROUND ART

The complement system is an important component of the innate immunedefense. A prerequisite for the complement system to exert its functionis its activation, which can occur through three different ways: theclassical, the lectin and the alternative pathways. Invading pathogenicmicroorganisms (e.g. bacteria, viruses, and fungi) can directly initiateeach distinct pathway before the adaptive immune response is developed.

The complement cascade, however, if inappropriately activated, can causea significant amount of inflammation, tissue damage, and other diseasestates such as the autoimmune diseases. Disease states implicating thecomplement system in inflammation and tissue damage include thefollowing: the intestinal inflammation of Crohn's disease (Ahrenstedt etal., 1990), thermal injury (burns, frostbite) (Gelfand et al, 1982;Demling et al., 1989), hemodialysis (Deppisch et al., 1990); Kojima etal., 1989), and post pump syndrome in cardiopulmonary bypass (Chenowethet al., 1981; Chenoweth et al., 1986; Salama et al., 1988), supposedlyit is involved in the development of fatal complication in sepsis (Hacket al., 1989) and causes tissue injury in animal models of autoimmunediseases. The complement system is also involved in hyperacute allograftand hyperacute xenograft rejection (Knechtle et al., 1985); Guttman,1974); Adachi et al., 1987). Complement activation during immunotherapywith recombinant IL-2 appears to cause the severe toxicity and sideeffects observed from IL-2 treatment (Thijs et al., 1990). Furtherdeleterious effects of improper activation or overactivation of thecomplement system is described e.g. in US Application No. 2002037915.

Based on increased incidence of infections in individuals with MBLdeficiency, there are indications in the art that the lectin pathway isassociated with the following diseases: HIV (increased susceptibility ofinfection), cystic fibrosis, systemic lupus erythematosus, rheumatoidarthritis, recurrent miscarriage, meningitis, cryptospirodiosis, chronichepatitis C (Dumestre-Perard, 2002) (as a disease modulator). Complementactivation, contributing to the inflammatory reaction upon observed inreperfusion injury is mediated through the lectin pathway (Monsinjon,2001, Collard, 2000). In a rat animal model, blockade of the lectinpathway protected the heart from ischemia-reperfusion by reducingneutrophil infiltration and attenuating proinflammatory gene expression.(Jordan, 2001)

It is of particular importance therefore to study key molecules of thecomplement system, their structure and function, therefore to obtainthese molecules or functional or folded fragments thereof in sufficientquantities and in a pure form to obtain appropriate research tools, todevelop assays for detecting said molecules and to find, design or raisemolecules for effecting the function of the complement system or forsupplying deficiencies of it and also treating decease conditionsassociated with irregular working of this system.

The activation of the complement system (like of other proteolyticcascades) results in the sequential activation of serine proteasezymogens. The first step in the lectin and the classical pathways is thebinding of a specific recognition molecule (MBL or C1q, respectively) toactivator structures, which is followed by the activation of associatedserine proteases (Gál, 2001).

Although the lectin pathway was discovered more than a decade ago(Kawasaki, 1989), there are many uncertainties concerning thecomposition of the activation complex and the substrate specificities ofthe MBL-associated serine proteases (MASPs). MBL is a member of thecollectin family of proteins and binds to specific carbohydrate arrayson the surface of various pathogens through C-type lectin domains(Turner, 1996). Up to date three MBL-associated serine proteases havebeen described. First, a single enzyme ‘MASP’ was identified andcharacterized as the enzyme, which is responsible for the initiation ofthe complement cascade (i.e. cleaving C2, C4 and possibly C3)(Matsushita, 1992/Ji, Y-H., 1993). Later it turned out that ‘MASP’ is infact a mixture of two proteases: MASP-1 and MASP-2 (Thiel, 1997). It wasdemonstrated, that the MBL-MASP-2 complex alone is sufficient forcomplement activation (Vorup-Jensen, 2000). This is a significantdifference from the C1 complex, where the coordinated action of twoserine proteases (C1r and C1s) leads to the activation of the complementsystem. Here, C1q is the recognition subunit of the complex, while C1rand C1s are highly specific serine proteases (with Mrs 86.5 kDa and 80kDa, respectively), which are responsible for the catalytic function ofC1. A specific feature of the C1r and C1s serine proteases is that theyform a distinct structural unit, the Ca²⁺-dependent C1s-C1r-C1r-C1stetramer, which makes possible the coordinated action of the two enzymeswithin the C1 complex. This tetramer associates with C1q to yield theheteropentameric C1 complex. C1r and C1s are present in the C1 complexin zymogen form, and become activated after C1q binds to an activator.The first enzymatic event during the activation process is theautoactivation of C1r. Activated C1r then activates zymogen C1s, whichin turn cleaves C4 and C2.

The role of MASP-1 in the MBL-MASPs complex remained unknown. It wasproposed, that MASP-1 could directly cleave C3 and thereby activatecomplement (Matsushita, 1995/Matsushita, 2000), but other laboratoriesdebated these results (Wong, 1999/Zhang, 1998). Recently, a novelprotease, MASP-3 has been isolated, however, its function is yet to beresolved (Dahl, 2001). Several lines of evidences suggest that there aredifferent MBL-MASPs complexes (Thielens, 2001/Dahl, 2001) and a largefraction of the total MASPs in serum is not complexed with MBL (Terai,1997/Thiel, 2000).

The MASPs together with C1r and C1s, form a family of proteases withidentical domain organization (Sim, 2000/Volanakis, 1998). In theseenzymes the first N-terminal CUB domain is followed by an EGF-likedomain and the second CUB domain. A tandem repeat of complement controlprotein modules (CCP1 and CCP2) precedes the C-terminal serine proteasedomain (SP). Upon activation an Arg-Ile bond is cleaved in the serineprotease domain of these zymogens.

Although the substrate specificities of MASP-1 and MASP-2 has beenstudied using natural and recombinant proteins, several importantquestions remained unanswered in the art. MASP-1 was shown to cleave C3and C2 (Matsushita, 2000), but this action may not be sufficient fordirect complement activation (Rossi, 2001). If the cleavage of C3 byMASP-1 proves to be insignificant then the field is still open to assessthe biological importance of MASP-1. This could be possibly accomplishedby identifying the range of its substrate specificity and the degree ofits specific activity and by finding a ‘better’ natural substrate thanC3. Previous studies showed that MASP-2 digested C2 and C4 efficiently,with rates similar to C1s, a classical pathway enzyme (Vorup-Jensen,2000/Rossi, 2001). However, the contribution of the individual domainsto the enzymatic properties of MASP-2 has not yet been determined. It isaccepted that C1-inhibitor reacts with both proteases (Matsushita,2000), but the rates of the reactions are unknown and the role ofanother inhibitory protein, alpha-2-macroglobulin, is rather debated(Wong, 1999/Rossi, 2001/Gulati, 2002). To sum up, differences infunction and biological role of MASP-1 and MASP-2 are unclear accordingto the art.

Similarly, before the creation of the subject invention, the role of theCCP and SP domains of C1r was unclear. The CCP repeat is about 60residues in length and is widespread among complement proteins. Itappeared to be likely that the CCP domains significantly contribute tothe specificity of the interaction and catalytic properties of the γBfragment. For example a recent structural model of (γB)₂ suggest a loosehead-to-tail assembly of the monomers, where the γ-chain (the two CCPmodules and the activation peptide) of one monomer interacts with theserine protease module of the other monomer (8).

Moreover, the autoactivation mechanism was unclear. Which domain isnecessary to autoactivation? At all, are the individual domains separatefolding units? Can folded fragments prepared? Are they active?

The answer to these questions and other problems of the art is animportant prerequisite of further research aiming at the treatment ofcomplement related disorders.

The main reason for these uncertainties concerning biological functionof these proteases is the lack of availability of active and/or nativerecombinant proteins in a sufficient quantity and purity. The functionalcharacterization has been retarded by the fact that their serumconcentration is very low (in the case of MASPs: [MASP-1]=6 μg/ml,[MASP-2]≈2 μg/ml) (Hajela, 2002) which rendered their isolationextremely difficult. Most MASP preparations obtained from serum wereusually cross-contaminated with other MASP species. Therefore, preciseexperiments could not be carried out in many cases. Also, though methodsfor determining in vivo levels of a mixture of MASPs have been known(e.g. U.S. Pat. No. 6,235,494 and U.S. Pat. No. 6,294,024) assessingMASP-1 and MASP-2 levels, differentiating one from the other, causedproblems according to the art.

Though JP 7238100 (Matsushita et al, 1995) is directed to a monoclonalantibody against human MASP, the defined character of such an antibodyis questionable since the publication does not make a distinctionbetween MASP-1 and MASP-2. In general, due to the lack of sufficientlypure preparations antibodies for native MASP proteins could not beproduced according to the art with a good reliability.

Similarly, a search for drugs, e.g. inhibitors, alleviating symptomsassociated with overactivity of the complement system had been greatlyhindered by the lack of availability of folded, possibly activefragments of key multidomain serine proteases of the complement system.

Also, in lack of a reliable and effective system for the recombinantpreparation of fragments of complement serine proteases associated withrecognition molecules of the complement system mutation studies andgenetic engineering of them were difficult and the outcome was difficultto interpret.

Last but not least methods of the art for producing MASP proteins andfragments were relatively expensive and cumbersome: cheaper and moreeffective methods were needed.

There have been many attempts in the art to prepare recombinant MASP-1and MASP-2. However, recombinant expression of the full-length MASPsprovided serious difficulties. In WO 02/6460 (Jensenius and Thiel, 2002)cloning and sequencing of MASP-2 is described. However, the protein wasnot recombinantly prepared. Vorup-Jensen et al. transiently expressedhuman MASPs in HE 293 cells, but their recombinant MASP-1 had unexpectedmolecular mass and showed no enzymatic activity (Vorup-Jensen, 2000).Though Vorup-Jensen et al. succeeded in preparing an active MASP2protein, this protein carried a His tag. Moreover, the preparationprocess was prolonged and complicated. Chen et al. tried to produce ratMASP-1 and MASP-2 in CHO cells, but the wild type proteases werecytotoxic to the cells and therefore only inactive mutants could beproduced (Chen, 2001). Rossi et al. expressed full-length human MASP-1and MASP-2 in a baculovirus insect cell system, but due to the very lowyield the proteases could not be purified to homogeneity (Rossi, 2001).

Rossi et al. (2001) expressed also CCP1-CCP2-SP fragments of MASP-1 andMASP-2 in the baculovirus insect cell system, which showed comparableenzymatic activities with the full-length molecules towards protein andester substrates. Said fragments were secreted by the insect cells.However, CCP-SP fragment of MASP-2 could not be successfully produced inthe baculovirus expression system (Rossi et al., 2001). Based on theseresults the authors concluded that the first CCP domain (CCP1) iscrucial for activity and propose that the smallest active fragment ofMASP proteins is the CCP1-CCP2-SP fragment. Moreover, expression in abaculovirus/insect cell system has obviously several disadvantages fromthe economic point of view that is relatively low yield, high costs andthe complexity of purification process. Moreover proteins secreted fromthe insect cells are subject to a possible protease attack.

Previously, the baculovirus-insect cell system was used to producerecombinant C1r and C1s and their fragments (14, 20, 21). The yield ofthe secreted recombinant proteins, however, was found to be low. Thecatalytic C-terminal γB fragment of C1r, consisting of the two CCPdomains followed by the activation peptide of the protease and theserine protease domain (B-chain), can be obtained by autolysis or bylimited proteolysis of extrinsic proteases (e.g. thermolysin) (6).However, no recombinant production of the γB fragment of C1r or C1s hadbeen disclosed before creation of the present invention. Also, nosmaller recombinant fragments of the C1r or C1s catalytic region hadbeen disclosed. In particular, no prokaryotic expression of C1r or C1sfragments had been suggested in the art.

To circumvent the problems outlined above the present inventors decidedto attempt recombinant expression of the catalytic fragments ofmultidomain serine proteases capable of binding to recognition moleculesof the complement cascade. In spite of the fact that these are complex,multi-domain proteins which are of mammalian origin, inventors decidedto choose a prokaryotic, in particular a bacterial system, moreparticularly an E. coli based system.

To the best of their knowledge, the present Inventors were the first toexpress and successfully refold multidomain serine proteases inbacterial hosts.

It has long been accepted in the art that the proteolytic γB fragmentsof C1r and C1s, which consist of three domains: two CCP modules and theserine protease domain, retain the catalytic activity of the entiremolecule both in terms of substrate specificity and catalytic efficiency(Villiers, 1985/Arlaud, 1986/Lacroix, 1989). Recent studies withrecombinant fragments of C1r and C1s reinforced this view (Rossi,1998/Lacroix, 2001). Since the MASP proteases share the same domainorganization with C1r and C1s, it seems plausible that the CCP1-CCP2-SPfragments mediate the catalytic activity of these enzymes, as well. Thisis supported by the recent studies of Rossi et al. who showed comparableenzymatic activities with the full-length molecules towards protein andester substrates and found that CCP1 domain is necessary to activity.

Applying a bacterial expression system, it is an object of the inventionto produce functionally active fragments of MASP-1, MASP-2, MASP-3, C1sand C1r, with a yield sufficient for structural and functionalcharacterization, as well as to provide a teaching to prepare thecorresponding fragments of C1s and MASP-3. Furthermore, a further objectof the invention is to create fragments from which the CCP domainspreceding the SP domain were successively deleted and to create therespective expression vectors. It is a further object of the inventionto functionally characterize the fragments.

Though in some bacterial systems, mainly in those providing lowexpression levels, the proteins could be expressed in a soluble form,overexpression systems are advantageous. A usual problem ofoverexpressing mammalian proteins in bacterial hosts is that theunfolded foreign protein forms inclusion bodies and has to be subjectedto renaturation, though this feature may turn out to be an advantage:the expressed proteins are protected against protease attack. Despite agreat number of proteins successfully renatured, protein refoldingremains a problem to be solved on a case-by-case basis [Rudolph andLilie, (1996)]. It is to be mentioned here that, as a matter of course,successful attempts are published and failures usually not.Nevertheless, even in the recent past protein refolding was consideredas an extremely difficult task. By now it is generally approved thatwhereas in vitro refolding of single domain proteins is likely to besuccessful, refolding of multidomain proteins remains a problem thesolution of which is far from being obvious (Fischer et al EPA 0 393 725A1, Ambrosius et al., EPA 0 500 108). Furthermore, it is also well-knownthat protein folding is usually started at the N-terminal of thepolypeptide chain. Therefore, if an N-terminal part of a protein isdeleted, its refolding is significantly encumbered. Moreover, to thebest of the Inventors' knowledge, multidomain serine proteases, inparticular of human origin, have not been prepared in a folded form in aprokaryotic expression system.

Being aware of the fact that larger molecules are more difficult to berefolded, but hoping that the presence of the N-terminal portion of themolecule may help inducing the folding process, Inventors attempted torenature the entire MASP molecules. After the failure of methods athand, Inventors used a variety of additives and removed them in astepwise manner which, of course, raises costs. In spite of this, nounambiguously positive results were obtained.

Surprisingly, inventors found that the C-terminal CCP1-CCP2-SP fragment(also named as γB-fragment after the nomenclature used for C1s and C1rproteins) of MASP proteins and of C1r and C1s proteins could berenatured with an improved renaturation method at a sufficient or, underpreferred conditions, at a high yield.

Applying this method for smaller fragments success or promising resultswere achieved. In view of former results of their own and of those ofRossi et al (2001) it is also surprising that inventors found theCCP2-SP and the SP fragments to be active.

The inventors also recognized that a particularly improved method can becarried out if a temperature below 10° C. and a pH above pH 8.7,preferably pH 9 or 10 is applied and, preferably, in the refoldingbuffer at least arginine is applied as a chaotropic agent.

In a similar expression system and renaturation method (which wasslightly modified by applying higher temperature and lower pH) inventorscould express and isolate recombinant fragments of C1r as well.

Having now large amounts of pure fragments available, Inventors couldprovide a detailed functional characterization of the proteins. Inparticular, Inventors found differences in substrate and inhibitorspecifities of MASP-1 and MASP-2, providing a basis for differentiallymeasuring their level in serum or in a biological sample.

The fragments obtained according to the invention can be usedadvantageously, e.g., in drug screening methods and for antibodyproduction. Results also suggest possible applicability of them, inparticular MASP-1, and of their inhibitors in medical treatments.

BRIEF DESCRIPTION OF THE INVENTION Methods

The invention relates to a recombinant method for the preparation of anunglycosylated folded C-terminal fragment of a multidomain serineprotease, comprising the following steps:

a bacterial expression vector for expressing a DNA insert encoding aC-terminal fragment of a multidomain serine protease is provided,

said protein fragment is produced in a bacterial host by using the saidvector and obtained in a folded form from the bacterial host.

Preferably, the multidomain serine protease encoded by the cDNA insertis a multidomain serine protease of the complement cascade (multidomaincomplement serine protease), wherein said serine protease is ofvertebrata, preferably mammalian, more preferably human origin.

Preferably, the multidomain serine protease comprises at least two CCPdomains and a serine protease domain, preferably in the following order:CCP1-CCP2-SP (which is the same domain structure as e.g. that of theγB-fragment of e.g. the C1r molecule).

More preferably a multidomain serine protease having the followingdomain structure CUB-EGF-CUB-CCP1-CCP2-SP and being capable of bindingto a recognition molecule of the complement pathway, e.g. to MBL or C1q.Preferably, the multidomain serine protease is selected from thefollowing: MASP-1, MASP-2, MASP-3, C1r and C1q.

Preferably, the multidomain serine protease C-terminal fragment encodedby the cDNA insert is a fragment of comprising one or more domainselected from the following domain types: CCP1, CCP2, SP.

More preferably, the C-terminal fragment has essentially a domainstructure of any of the following: CCP1-CCP2-SP, CCP2-SP, SP.

The C-terminal fragment is preferably selected from any of thefollowing: MASP-1 CCP1-CCP2-SP fragment, MASP-2 CCP1-CCP2-SP fragment,MASP-2 CCP2-SP fragment or MASP-2 SP fragment, MASP-3 CCP1-CCP2-SPfragment, MASP-3 CCP2-SP fragment or MASP-3 SP fragment, C1rCCP1-CCP2-SP fragment (i.e. γB-fragment), C1r CCP2-SP fragment or C1r SPfragment, C1s CCP 1-CCP2-SP fragment (i.e. γB-fragment), C1s CCP2-SPfragment or C1s SP fragment.

As will be understood, the fragments of the invention may comprise notonly “whole” domains but also parts of domains provided that thefragment comprises at least one domain which is in the folded form.Preferably, the fragments of the invention comprise only folded domainsbesides of course, if desired, further useful sequences, e.g. tagsequences, as detailed below.

In a preferred embodiment the fragment of the invention comprises on itsN-terminal a tag sequence the coding sequence of which increases theefficiency of bacterial protein expression.

Said tag sequence is expediently a sequence suitable for the promoterused for expression, preferably a T7-tag sequence comprising preferably3 or 4 amino acids, preferably of the sequence: Ala-Ser-Met-(Thr). SEQID NO:19, SEQ ID NO:20.

The fragment of the invention may also lack on its N-terminal a tagsequence the coding sequence of which increases the efficiency ofbacterial protein expression due to a proteolytic cleavage (e.g.autolytic cleavage), e.g. it is a MASP-2 CCP1-CCP2-SP fragment startingwith Ile²⁹¹ at its N-terminus.

In a further preferred embodiment said fragment comprises mutation.

In a preferred embodiment the bacterial host is E. coli and theexpression vector is a vector capable of expressing, preferablyoverexpressing foreign genes in E. coli.

In a highly preferred embodiment E. coli BL21 strain is used, thepromoter is a T7 promoter, and the vector is a pET vector or a pSE-420vector.

In overexpression systems the expressed foreign proteins most often forminclusion bodies. Nevertheless, an embodiment wherein the proteinfragments are expressed in a soluble form is also within the claimedscope of the invention.

Preferably, the step of obtaining the C-terminal fragment in a foldedform comprises the following steps:

i) the inclusion bodies formed in the bacterial host are isolatedii) the protein fragment molecules are renatured from the inclusionbodies,iii) optionally, the renatured protein fragment molecules are furtherpurified.

Purification is carried out preferably by ion exchange chromatography,more preferably by a combination of anion and cation exchangechromatography. In this case preferably a higher than 70%, morepreferably a higher than 80%, even more preferably a higher than 90%purity is achieved, as detectable by SDS-PAGE.

Refolding of MASP-Fragments

In a preferred embodiment the protein fragment is MASP-fragment,preferably any of the MASP-fragments as defined above.

M′m.3. For the preparation of MASP protein fragments, in the preparationmethod step c) above comprises the following steps:

i) solubilization of the inclusion bodies,ii) diluting the solubilized protein fragments and transferring theminto a refolding buffer,iii) allowing the protein fragments to refold at

-   -   a pH higher than 7, preferably above pH 8.5, more preferably        between 8.5 and 10.5 and    -   a temperature between 0 and 15° C.,    -   in an environment suitable for mildly oxidizing cysteine        residues into cystines,        iv) transferring the refolded protein fragments into an        appropriate buffer.

In step i) solubilization can be carried out in an appropriatesolubilization agent, e.g. GuHCl or urea, preferably in a buffercomprising 6M GuHCl, any suitable reducing agent e.g. β-mercaptoethanolor DTT, e.g. 100 mM DTT, and optionally a buffering agent, e.g. 0.1 MTris-HCl (pH 8.3), e.g. at room temperature or somewhat lower. In orderto avoid oxidation of the reducing agents (in particular if DTT isused), long incubation periods should be avoided.

The protein concentration in the solution is preferably 0.5 to 25 mg/ml,more preferably 1 to 20 or 2 to 10 mg/ml, even more preferably 3 to 7,e.g. about 5 mg/ml.

In step ii) the solubilized proteins are preferably diluted directlyinto the refolding buffer. The dilution is at least 10 fold, preferablyat least 100 fold, more preferably 200-600 fold, even more preferablyabout 400 fold. The protein can be added dropwise or in relatively smallportions.

Prior dilution the refolding buffer is cooled below 10° C., preferablybelow 5° C., more preferably to about 0° C. Preferably, the buffer isdegassed before adding the solubilized protein.

In step iii), during refolding, the refolding buffer comprises a redoxsystem, e.g. cysteine/cystine system or a glutathione system wherein theratio of the reduced and oxidized forms is set to provide an mildlyreductive environment, e.g. wherein the reduced/oxidized ratio is 10:1to 1:1. Preferably, glutathione is used wherein the ratio of the reducedand oxidized forms is about 3 to 1, e.g. 3 mM and 1 mM, respectively.

The refolding buffer further comprises a chaotropic agent in anappropriate concentration, e.g. arginine (0.5 to 1.0 M). In case ofMASP-2, GuHCl (0.5 to 2.0 M) can be used. A mixture of arginine andGuHC1 is also applicable within the concentration ranges given above(preferably at most 3.0 M together), and being aware of this fact theskilled person will be able to set the appropriate ratio for eachprotein.

Refolding is carried out at a temperature between 0 to 15° C.,preferably between 0 to 10° C., more preferably between 4 to 8° C. In ahighly preferred embodiment the refolding is carried out at 6° C.

The protein fragments may refold very fast; nevertheless, they should beallowed to refold for a while to reduce the number of aggregates andmisfolded molecules, e.g. at least a few minutes or preferably at leasthours e.g. at least 2, 3 or 4 hours or overnight. The proteins may beallowed to refold for longer, e.g. one or two days or even more.Duration is limited by usual factors influencing limits of storage ofprotein solutions.

Preferably, the refolding buffer comprises a protease inhibitor, e.g.EDTA or one or more other known protease inhibitor and any appropriatebuffering agent, e.g. 50 mM Tris.

Thus, in a preferred embodiment of the method

i) solubilization is carried out in the presence of 5 to 6 M GuHCl in asuitable solubilization buffer,ii) the solubilized proteins are diluted 200 to 600 fold into therefolding buffer previously cooled to a temperature between 0 to 10° C.,iii) the refolding buffer comprises a chaotropic agent, preferablyarginine; reduced and oxidized glutathiones in a ratio of 10:1 to 1:1;and preferably a buffering agent and one or more protease inhibitor(s);and refolding is carried out at a temperature between 0 to 10° C.iv) the refolded proteins are dialyzed into a buffer of a pH above thepI of the protein fragment.

In a preferred embodiment the MASP protein fragment is MASP-1CCP1-CCP2-SP fragment, MASP-2 CCP1-CCP2-SP fragment or MASP-2 CCP2-SPfragment and the pH of the refolding buffer is between pH 8.5 and 9.5,preferably about pH 9.0.

In a further preferred embodiment the MASP protein fragment is MASP-2 SPfragment and the pH is between pH 9.5 to 10.5, preferably pH 10.

In a further preferred embodiment the MASP protein fragment is MASP-2fragment and in the refolding buffer the chaotropic agent is 0.5 to 2 MGuHCl or at least 0.5 M of a mixture of maximum 2 M GuHCl and maximum 1M arginine.

Refolding of C1r Fragments

In a further preferred embodiment the protein fragment is C1 serineprotease fragment, e.g. a C1s or a C1r fragment, preferably any of theC1r fragments as defined above.

Preferably, for the preparation of C1r protein fragments, in thepreparation method step c) comprises the following steps:

i) solubilization of the inclusion bodies,ii) diluting the solubilized protein fragments and transferring theminto a refolding buffer,iii) allowing the protein fragments to refold at

-   -   a pH higher than 5, preferably between 7 and pH 8.5 and    -   a temperature between 0 and 15° C.,    -   in an environment suitable for mildly oxidizing cysteine        residues into cystines        iv) transferring the refolded protein fragments into an        appropriate buffer.

In step i) solubilization can be carried out in an appropriatesolubilization agent, e.g. GuHCl or urea, preferably in a buffercomprising 6M GuHCl, any suitable reducing agent e.g. β-mercaptoethanolor DTT, e.g. 100 mM DTT, and optionally a buffering agent, e.g. 0.1 MTris-HCl (pH 8.3), e.g. at room temperature or somewhat lower. In orderto avoid oxidation of the reducing agents (in particular if DTT isused), long incubation periods should be avoided.

The protein concentration in the solution is preferably 0.5 to 25 mg/ml,more preferably 1 to 20 or 2 to 10 mg/ml, even more preferably 3 to 7,e.g. about 5 mg/ml.

In step ii) the solubilized proteins are preferably diluted directlyinto the refolding buffer. The dilution is at least 10 fold, preferablyat least 100 fold, more preferably 200-600 fold, even more preferablyabout 400 fold. The protein can be added dropwise or in relatively smallportions.

In step iii), during refolding, the refolding buffer comprises a redoxsystem, e.g. cysteinelcystine system or a glutathione system wherein theratio of the reduced and oxidized forms is set to provide an mildlyreductive environment, e.g. wherein the reduced/oxidized ratio is 10:1to 1:1. Preferably, glutathione is used wherein the ratio of the reducedand oxidized forms is about 3 to 1, e.g. 3 mM and 1 mM, respectively.

The refolding buffer further comprises a chaotropic agent in anappropriate concentration, preferably GuHCl (preferably 2.0 M).

Refolding is carried out at a temperature between 0 to 15° C.,preferably between 4 to 15° C.

The protein fragments may refold very fast; nevertheless, they should beallowed to refold for a while to reduce the number of aggregates andmisfolded molecules, e.g. at least a few minutes or preferably at leasthours e.g. at least 2, 3 or 4 hours or overnight. The proteins may beallowed to refold for longer, e.g. one or two days or even more.Duration is limited by usual factors influencing limits of storage ofprotein solutions.

Preferably, the refolding buffer comprises a protease inhibitor, e.g.EDTA or one or more other known protease inhibitor and any appropriatebuffering agent, e.g. 50 mM Tris.

Bacterial Expression Vectors

In a yet further aspect of the invention a prokaryotic expression vectoris provided, said vector comprising a DNA insert encoding a C-terminalfragment of a multidomain serine protease and means for expressing saidfragment in a bacterial host.

Preferably, the prokaryotic expression vector is a bacterial vector andsaid DNA insert is a cDNA insert.

Preferably, the cDNA insert encodes a fragment comprising one or moredomain selected from the following domain types: CCP1, CCP2, SP.

Preferably, the said cDNA insert encodes a fragment of a multidomainserine protease of the complement cascade (multidomain complement serineprotease), more preferably a multidomain serine protease capable ofbinding to a recognition molecule of the complement pathway, e.g. to MBLor C1q, preferably a multidomain serine protease selected from thefollowing: MASP-1, MASP-2, MASP-3, C1r and C1q.

Preferably, the c-DNA insert encodes a protein fragment havingessentially a domain structure of any of the following: CCP1-CCP2-SP,CCP2-SP, SP.

Preferably, the C-terminal fragment is selected from any of thefollowing: MASP-1 CCP1-CCP2-SP fragment, MASP-2 CCP1-CCP2-SP fragment,MASP-2 CCP2-SP fragment or MASP-2 SP fragment, MASP-3 CCP1-CCP2-SPfragment, MASP-3 CCP2-SP fragment or MASP-3 SP fragment, C1rCCP1-CCP2-SP fragment (i.e. γB-fragment), C1r CCP2-SP fragment or C1r SPfragment, C1s CCP1-CCP2-SP fragment (i.e. γB-fragment), C1s CCP2-SPfragment or C is SP fragment.

In a preferred embodiment, the cDNA insert comprises a sequence encodinga further amino acid sequence operably linked to the sequence encodingthe protein fragment. Such further sequence can be e.g. a sequenceincreasing the efficiency of bacterial protein expression, or a sequenceencoding a tag sequence, e.g. an N-terminal tag sequence.

Said tag sequence is expediently a sequence suitable for the promoterused for expression, preferably a T7-tag sequence comprising preferably3 or 4 amino acids, preferably of the sequence: Ala-Ser-Met-(Thr). SEQID NO:19, SEQ ID NO:20.

In a further preferred embodiment said fragment comprises mutation.

Any vector as described above wherein the means for expressing the DNAinsert sequence encoding any of the protein fragments as disclosedabove, operably linked to a promoter, preferably an inducibleoverexpressing promoter capable of driving expression in a suitablebacterial host. Preferably, the bacterial host is E. coli, morepreferably an E. coli strain suitable for overexpression, e.g. the BL21strain, and the promoter is a promoter driving overexpression, e.g. theT7 promoter. The vector advantageously is a multicopy vector carrying aselectable marker and a suitable cloning site, e.g. the vector is a pETvector or a pSE-420 vector.

Protein Fragments

In an aspect the invention relates to a C-terminal fragment of amultidomain serine protease of the complement cascade (multidomaincomplement serine protease), obtained by the method of any of M, whereinsaid fragment is unglycosylated and folded.

In a preferred embodiment, the multidomain serine protease is a serineprotease capable of binding to a recognition molecule of the complementpathway, e.g. MBL or C1q.

The multidomain serine protease according to the invention is ofvertebrata, preferably mammalian, more preferably human origin.

In an aspect the invention relates to an unglycosylated foldedC-terminal fragment of a multidomain serine protease comprising at leasttwo CCP domains and a serine protease domain, preferably in thefollowing order: CCP1-CCP2-SP, which is the same domain structure ase.g. that of the γB-fragment of e.g. the C1r molecule. Preferably, theserine protease has the following domain structureCUB-EGF-CUB-CCP1-CCP2-SP.

Preferably, the multidomain serine protease is a MASP.

In a further embodiment, the multidomain serine protease is a serineprotease of the C1 complex.

Preferably, the C-terminal fragment comprises one or more of any of thefollowing domains: CCP1, CCP2, SP, more preferably has essentially adomain structure of any of the following: CCP1-CCP2-SP, CCP2-SP, SP.

Preferably, the C-terminal fragment is selected from any of thefollowing: MASP-1 CCP1-CCP2-SP fragment, MASP-2 CCP1-CCP2-SP fragment,MASP-2 CCP2-SP fragment or MASP-2 SP fragment, MASP-3 CCP1-CCP2-SPfragment, MASP-3 CCP2-SP fragment or MASP-3 SP fragment, C1rCCP1-CCP2-SP fragment (i.e. γB-fragment), C1r CCP2-SP fragment or C1r SPfragment, C1s CCP1-CCP2-SP fragment (i.e. γB-fragment), C1s CCP2SPfragment or C1s SP fragment.

As will be understood, the fragments of the invention may comprise notonly “whole” domains but also parts of domains provided that thefragment comprises at least one domain which is in the folded form.

In an embodiment the fragment of the invention comprises an additionalamino acid sequence.

In a preferred embodiment the fragment of the invention comprises on itsN-terminal a tag sequence the coding sequence of which increases theefficiency of bacterial protein expression.

Said tag sequence is expediently a sequence suitable for the promoterused for expression, preferably a T7-tag sequence comprising preferably3 or 4 amino acids, preferably of the sequence: Ala-Ser-Met-(Thr).

The fragment of the invention may also lack on its N-terminal a tagsequence the coding sequence of which increases the efficiency ofbacterial protein expression due to a proteolytic cleavage (e.g.autolytic cleavage). For example, the fragment can be a MASP-2CCP1-CCP2-SP fragment starting with Ile²⁹¹ at its N-terminus.

Any fragment, however, which carries an evidence of having been producedby a nonbacterial expression system is of course excluded from the scopeof the invention. For example fragments having a secretion signalsequence, either mammalian or a signal sequence operable in insectcells, or a part thereof, or additional amino acids obviously used forlinking the fragments of the multidomain serine protease to a suchsequence are not claimed according to the invention. For example, MASP-2CCP1-CCP2-SP fragments carrying such sequences are excluded. Fragments,which are glycosilated, are not covered by the present invention, aswell.

In a further preferred embodiment said fragment of the invention isautoactivated or capable of being autoactivated.

In a further preferred embodiment said fragment comprises mutation.

The mutated fragment can be inactivated or can be incapable ofautoactivating. Thereby said mutants are proof against autoactivationand autodegradation.

In a highly preferred embodiment the activation site of the SP-domain,e.g. the Arg-Ile bond of the activation site is mutated, preferably toGln-Ile. It is preferred, if the mutants can be activated in a regulatedway, e.g. by thermolysin.

According to a further possibility, the active Ser is mutated, e.g. toGly, Ala, Thr, Val, etc., preferably to Ala.

As will be understood, any amino acid or a group of amino acids can bemutated in the fragments by using the expression and purification systemof the invention. Furthermore, deletion mutants can be created, e.g.C-terminal deletion mutants which may be inactive, as well.

In a further aspect, the invention also relates to folded, functionalMASP-2 CCP2-SP fragments and MASP-2 SP fragments.

Uses

In a further aspect of the invention the following uses are provided:

Use of any of the fragments of the invention for raising antibodies.Preferably, said antibodies are suitable for detecting foldedmultidomain complement serine proteases, e.g. folded MASP-1, MASP-2 orMASP-3, more preferably MASP-1 or MASP-2 or measuring folded MASP-1 orMASP-2 or MASP-3, more preferably MASP-1 or MASP-2 level in a biologicalsample.

Use of any of the fragments of the invention as a standard or a controlin assessing the level or activity of a multidomain complement serineprotease in a biological sample.

The level of a multidomain complement serine protease, a MASP, C1r orC1s, can be measured by a labeled monoclonal antibody. Many ways of thisis known, e.g. ELISA, RIA, DELFIA etc. If the antibody is bound to anepitope on a CCP1, CCP2 or an SP domain, a suitable fragment of the saidprotease according to the invention can be used as a control or standardprovided that said fragment comprises the domain carrying said epitope.

If activity of a multidomain complement serine protease is assayed in abiological sample on a substrate of said protease, e.g. a substrateobtained by screening according to the invention, in such an assay anappropriate fragment of said protease according to the invention can beused as a standard or a control.

Use of the fragments of the invention for screening substrates orinhibitors of MASP-1 or MASP-2 or MASP-3, more preferably MASP-1 orMASP-2. In a preferred embodiment oligopeptides are screened, e.g.peptides comprising Arg and/or Lys residue(s).

Suitable antibodies can inhibit binding of the fragment to theirsubstrates. Such antibodies, preferably monoclonal antibodies can beprepared and then selected by screening according to the invention.Moreover, antibodies inhibiting dimerization of C1r molecules can bedevised or prepared (raised and selected). Thereby the activation of theclassical pathway could be inhibited selectively.

Thus, the invention also relates to antibodies prepared by using theprotein fragments of the invention.

The fragments of the invention can be used to prepare composite proteinmolecules functioning as whole MASP molecules, provided that the lackingN-terminal fragment of the MASP in question, comprising the rest ofdomains or a part of them, is provided in a native form and bound to theC-terminal portion of a fragment according to the invention. Thereby atool for studying domain structure and the role of individual domainscan be prepared.

Thus, the fragments of the invention can be used as research tools in avariety of fields related to complement research as demonstrated herein.

Furthermore, the invention relates to the use of any of the MASP-1fragments of the invention for the preparation of a pharmaceuticalcomposition for inducing blood coagulation and the use of an inhibitorof MASP-1 for reducing blood coagulation. Preferably, said inhibitor isM or C1-inhibitor.

Assay Methods

According to a further aspect the invention relates to the followingassay methods:

An assay method for measuring the level of a multidomain complementserine protease in a biological sample, wherein the presence of saidserine protease is quantitatively detected in the sample by a labeledmonoclonal antibody and the obtained signal is compared with a signalobtained for a control sample comprising a respective complementprotease fragment according the invention.

An assay method for measuring the activity of a multidomain complementserine protease in a biological sample, wherein the activity on asubstrate of said protease is measured, e.g. a substrate obtained byscreening according to the invention, and an appropriate fragment ofsaid protease according to the invention is used as a standard or acontrol provided that it has the same specific activity as therespective protease or the ratio of the activities of the nativeprotease and the fragment is known. Preferably, in this assay method aCCP1-CCP2-SP fragment of said protease is used.

An assay method for assessing MASP-1 and MASP-2 levels in a sample ofbiological origin, said method comprising

-   -   monitoring C2 cleavage and C4 cleavage by MASP proteins in        aliquots of the sample whereas, if desired, other complement        pathways are blocked,    -   considering C4 conversion as a result of MASP 2 activity and C2        conversion as a result of MASP-1 and MASP-2 activity together    -   calculating MASP-1 and MASP-2 levels using either known specific        activity values of said proteins or MASP-1 and MASP-2        CCP1-CCP2-SP fragments, respectively, as inner standards.

An assay method for assessing MASP-1 and MASP-2 levels in a sample ofbiological origin, said method comprising

-   -   monitoring C2 cleavage in the sample and considering C2        conversion as a result of MASP-1 and MASP-2 activity together,    -   adding a calculated amount of α2M to the sample to inhibit        MASP-1 activity but leaving MASP-2 activity unchanged or        changing it to a negligible or a calculable extent,    -   monitoring C2 activity in the sample comprising α2M,    -   calculating MASP-1 and MASP-2 levels using either known specific        activity values of said proteins or MASP-1 and MASP-2        CCP1-CCP2-SP fragments, respectively, as inner standards.

Methods for blocking the classic and the alternative pathways of thecomplement cascade are known, see e.g. U.S. Pat. No. 6,297,024.

A diagnostic kit for carrying out any of the assay methods of theinvention said kit comprising a fragment of the invention is alsoprovided.

Treatment

The invention also relates to a method for inducing fibrin formationfrom fibrinogen or blood coagulation comprising adding any of the MASP-1fragments to a sample or administering them to a subject.

The invention also relates to a method for treating patients deficientin MASP-1 or in need of MASP-1 by administering a MASP-1 CCP1-CCP2-SPfragment to the patient.

The invention further relates to a method of reducing the activity of ahuman MASP-1 serine protease, comprising contacting said MASP-1 with α2Min at least a stoichiometrically significant quantity.

Moreover, the invention relates to a method for treatment of a patientin need of inhibiting complement activity, comprising administering anyinhibitor of the respective complement pathway prepared or screened asdescribed above. Preferably, the invention further relates to a methodfor treatment of a patient in need of inhibiting complement activityexerted through the lectin pathway, which rises in complement relateddiseases, said treatment comprising the administration of any inhibitorof the lectin complement pathway, preferably an inhibitor of MASP-2,prepared or screened as described above. Preferably, the treatedcondition is reperfusion injury.

DEFINITIONS AND ABBREVIATIONS

A “multidomain complement serine protease” is understood herein as amultidomain serine protease of the complement cascade.

A “supramolecular complex initiating the complement cascade” is meantherein as a supramolecular protein complex capable of binding toactivator structures and activate the corresponding pathway of thecomplement system. Such complexes e.g. are the MBL/MASP complex or theC1 complex.

A “recognition molecule of the complement system” is a molecule capableof binding to activator structures triggering the complement cascade.Such molecules are e.g. MBL and C1q which are parts of supramolecularcomplex initiating the complement cascade.

A “MASP molecule” is understood as an MBL-associated serine proteasehaving a domain structure consisting of at least two complement controlprotein (CCP) and one serine protease (SP) domain or module. The domainstructure of a MASP molecule is preferably the following:CUB-EGF-CUB-CCP1-CCP2-SP.

A “CCP domain” (complement control protein domain) is a domain of about50 to 70, preferably about 60 amino acids having a compact hydrophobecore comprising at least 5 antiparallel β-sheets. A CCP domain comprises4 conserved cystein residues which form disulfide bridges according tothe following pattern: 1-3, 2-4.

An SP domain (“serine protease”) of a multidomain complement serineprotease, in particular a MASP, C1r or C1s is in general characterizedby a chymotrypsin-like fold, i.e. which has two beta-barrel domains, andeach of them contains 6 beta-sheets arranged as antiparallel sheets. Theactive site residues are far apart in the primary sequence but arebrought together in a crevice.

The terms “module” and “domain” have the same meaning throughout thedescription. Domain boundaries of domains of multidomain complementserine proteases as mentioned herein are basically described in theComplement Factsbook [By Morley, B., Published By Academic Press, Inc.,(2000)] which is incorporated herein by reference. Nevertheless, aprotein fragment is considered herein as comprising an essentially wholedomain if the polypeptide portion of said domain, which is at leastessentially capable of folding to its 3 dimensional structure orcomprises sufficient amino acids so as to have a definite 3 dimensionalstructure.

A protein “fragment” of a multidomain complement serine protease ismeant herein as a portion of a polypeptide having a sequence homologousto the sequence of the corresponding portion of a known variant of saidmolecule, wherein the level of homology is at least 50%, preferably atleast 60 or 70, even more preferably 80, 85, 90, 93, 95, 96, 97 or 98%.

The amino acid positions in protein fragments are numbered according tothe numbering of the multidomain complement serine protease.

A C-terminal fragment of a multidomain complement serine protease is afragment comprising essentially the C-terminal amino acid sequence ofthe said protease or a fragment the C-terminal amino acid of which is anamino acid the position of which, considering the numbering of theserine protease, is not farther then 50, preferably 40, 30, 20, 15 orhighly preferably 10 or 5 amino acid positions from the originalC-terminal position of the said multidomain complement serine protease.

A “folded fragment” of a multidomain complement serine protease, e.g. aserine protease of a supramolecular complex initiating the complementcascade, e.g. a MASP molecule, a C1r or a C1s molecule, is meant hereinas a fragment comprising at least one domain, corresponding to a domainof a known MASP or C1r or a C1s molecule, said domain having essentiallya native tertiary structure as detected by any method known to besuitable for that purpose in the art, e.g. SDS-PAGE, activity, or anymethod for detecting a function of the molecule, and spectroscopicmethods e.g. fluorescence methods, CD-spectroscopy or proteincrystallography. Preferably, all the domains in the folded fragment arefolded. As a matter of course a folded fragment may contain peptidesegments which constitute a part of another domain of the MASP moleculeor additional sequences or amino acids (e.g. tags) provided that theabove criteria are fulfilled.

The abbreviation α2M is for α-2-macroglobulin, whereas C1, C2, C3, C4and C1i or C1 inhibitor are the usual abbreviations of components of thecomplement cascade.

Other abbreviations, terms and expressions are used herein as common inthe art.

Sequences of human MASP as well as C1r and C1s proteins are well knownin the art from various sources. Such sequences are given e.g. in theEntrez Protein database, e.g. at the following accession numbers:

MASP-1

NP_(—)001870 (isoform 1)NP_(—)624302 (isoform 2)XP_(—)193834 (mouse)NP_(—)071593 (rat) etc

MASP-2

000187 (human, precursor)NP_(—)006601 (human, isoform 1))

MASP-3

AAN39851 (rat)BAB69688 (mouse)

C1r

P00736 (human, precursor)NP_(—)001724 (human)

C1s

P09871 (human, precursor)NP_(—)659187 (mouse)

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: A schematic representation of pathways of complement activation.Unclarified parts standing in the focus of the attention of the presentstudy are indicated in light gray.

FIG. 2: Modular structure of proteins known to be associated with MBL.Five non-enzymatic domains can be found at the N-terminus of the MASPsand a serine protease domain at the C-terminus, which are cleaved uponactivation.

FIG. 3: Expression of the MASP-1 and MASP-2 fragments. Expression wascarried out as described in the Materials and Methods section.

FIG. 4: Coomassie stained SDS-PAGE of the purified MASP-1 and MASP-2fragments (MASP-1γB, MASP-2γB, MASP-2 CCP2-SP, MASP-2 SP). Followingrenaturation the fragments were purified using ion exchange andhydrophobic interaction chromatographies on Mono-S, Mono-Q and HIC PEcolumns. The MASP-2 fragments became activated during the purificationprocess indicating the capability to autoactivate. MASP-1γB was fullyactivated before the chromatography steps and co-purified with a majorcontaminant. N-terminal amino acid sequencing yielded the expectedsequences.

FIG. 5: C2, live C3, dead C3, C4 cleavage activities of the MASP-1 andMASP-2 fragments. Purified fragments were incubated with purified C2(A), live C3, C3(NH₃) (B) and C4 (C) for 0 to 40 minutes. Samplealiquots were taken to monitor the reaction. The samples were run onSDS-PAGE and the decrease of the cleavable chains was analyzeddensitometrically. Upon preincubation of the MASP fragments withC1-inhibitor no cleavages could be observed.

FIG. 6: Radiogram of an SDS-PAGE run under reducing conditions of theindicated inhibitors with the MASP-1 and MASP-2 fragments (MASP-1γB,MASP-2γB, MASP-2 CCP2-SP, MASP-2 SP)

FIG. 7: The percentage of reacted peptides (% decrease of peak area,y-axis) of a given P1 site (Lys or Arg) during a time period given inminutes (x-axis). Peaks are normed.

FIG. 8: Agarose gel of amplified MASP-3 fragment

FIG. 9: Modular structure of human C1r Recombinant fragments of thecatalytic region used in this study were CCP1-CCP2-SP, CCP2-SP, and SP.The γB fragment that can be obtained from the entire C1r molecule bylimited proteolysis and our recombinant CCP1-CCP2-SP fragment have thesame domain structure. The S654A catalytic site- and the R463Qactivation site mutants are also presented.

FIG. 10. SDS-PAGE of the renatured and purified C1r fragments A:Wild-type C1r fragments, non reducing conditions: (lane 1) CCP1-CCP2-SP,(lane 2) CCP2-SP, (lane 3) SP; reducing conditions: (lane 4)CCP1-CCP2-SP, (lane 5) CCP2-SP, (lane 6) SP. Proteins were analysed on a12.5% polyacrylamide gel. The enzymes are fully activated afterpurification. B: R463Q mutant zymogen fragments and their thermolysinactivated two-chain forms (10 U thermolysin/mg C1r fragment, 2 h, 30°C.), reducing conditions: (lane 1, 2) R463Q CCP1-CCP2-SP, (lane 3, 4)R463Q CCP2-SP, lane (5, 6) R463Q SP, zymogen and activated forms,respectively, (lane 7) thermolysin.

FIG. 11. Relative molecular weights of C1r fragments vs. pH determinedby gel filtration chromatography SP (λ), CCP2-SP (τ), and CCP1-CCP2-SP(v). CCP1-CCP2-SP shows a dimer-monomer transition with a midpoint at pH5.5. Molecular weights were determined on a Superose-12 column using aprotein standard series at every pH, at 25° C., as described inMaterials and methods.

FIG. 12. DSC melting curves of the C1r fragments SP (______) , CCP2-SP(- - - -) and CCP1-CCP2-SP (. . . . . .) in 20 mM Tris pH 8.3 buffercontaining 145 mM NaCl. The protein concentration was 0.1 mg/ml. Aheating rate of 1° C./min was used.

FIG. 13. Proenzyme C1s cleavage by C1r CCP1-CCP2-SP The figure shows atypical experiment which was carried out in 20 mM Tris pH 7.4 containing145 mM NaCl with an enzyme:substrate ratio of 1:50 at 30° C. Thegenerated active C I s molecules were monitored through theiresterolytic activity on Z-Lys-S-Bzl substrate.

FIG. 14. Cleavage of CCP2-SP S654A zymogen mutant by wild-type CCP2-SPThe reaction was performed in 20 mM Tris, 145 mM NaCl, pH 8.3 at 37° C.An enzyme:substrate ratio of 1:15 was used. The activation process wasfollowed by 12.5% SDS-PAGE under reducing conditions. Reaction times forthe appropriate lanes are indicated. After cleavage the CCP2 and SPdomains are separated under reducing conditions.

FIG. 15. Zymographic detection of MASP-1 and MASP-2 on gelatine; photoof the PAGE gel. White band shows that MASP-1 has digested aconsiderable amount of gelatine whereas MASP-2, having a significantlynarrower substrate specificity, has not.

DETAILED DESCRIPTION OF THE INVENTION

Below the invention is further explained via specific examples. Theskilled person will understand that many variations based on the sameinventive idea can be carried out, said variations being thereforewithin the scope of the invention.

1. Materials and Methods Construction of Recombinant Plasmids for theExpression of the MASP-1, MASP-2 and MASP-3 Fragments

Coding sequences of MASP-1 and MASP-2 fragments were obtained by PCRstarting from bacterial vectors comprising full length cDNAs of thehuman liver MASP proteins (the vectors were a kind gift of Dr. Schwable,W, Institut fur Hygiene, University of Innsbruck, Austria). The cDNAsare also available from human liver cDNA library and are disclosed e.g.by Takada F et al. (1993) and Sato T et al. (1994) (MASP-1, Swiss-Protaccession No: P48740) and by Thiel S et al. (1997) and Stover C. M. etal. (MASP-2, Swiss-Prot accession No: O00187).

For all recombinant constructs the pET-17b expression vector wasdigested with NheI and EcoRI restriction endonucleases and PCR productswith identical sticky ends were ligated into the plasmid. In the case ofthe MASP-1 CCP1-CCP2-SP construct the following forward and reverseprimers were used: GCGGCTAGCATGACTGGTAATGAGTGCCCAGAGCTA, SEQ ID NO:1,and GCGGAATTCTCAGTTCCTCACTCCGGTGACCCT, SEQ ID NO:2, (the NheI and EcoRIrestriction sites are highlighted).

Preceding the MASP-1 sequence the forward primer contained the codon offour amino acids (Ala-Ser-Met-Thr) of the T7-Tag sequence, whichincreased the efficiency of the recombinant protein expression. As aresult of this the N-terminus of the recombinant MASP-1 contained thefour extra amino acids and the MASP-1 sequence began with Gly²⁹⁸ andended with Asn⁶⁹⁹. The same strategy was followed upon constructing theMASP-2 fragments. The reverse primer for the three MASP-2 constructs wasthe same: GCGGAATTCTTAAAAATCACTAATTATGTTCTCG, SEQ ID NO:3, therefore allrecombinant fragment ended with Phe⁶⁸⁶. The forward primers for MASP-2CCP1-CCP2-SP, CCP2-SP and SP were: GCGGCTAGCATGACTGGTTGGAAGATCCACTACACG,SEQ ID NO:4, GCGGCTAGCATGACTATTGTTGACTGTGGCCCTCCTG, SEQ ID NO:5, andGCGGCTAGCATGACTCCTGTTTGTGGACTATCAGCC, SEQ ID NO:6, respectively.

The recombinant CCP1-CCP2-SP construct contains the Ala-Ser-Mettripeptide before the Thr²⁸⁷ amino acid of MASP-2 at the N-terminus. Inthe case of the CCP2-SP and SP fragments the Ala-Ser-Met-Thr extratetrapeptide is followed by Ile³⁶³ and Pro⁴³² of MASP-2, respectively.

For cloning MASP-3 only two new primer had to be designed, since itsfirst five domains is identical with those of MASP-1 (i.e. only theserine-protease domain is different).

Reverse primer to MASP-3 SP domain:

GCGGAATTCTCACCGTTCCACCTGGGGCTCCAC,, SEQ ID NO:7

This primer comprises the EcoRI cleavage site to facilitate insertingthe PCR-product into an expression vector. The recombinant proteins willthus end with a C-terminal Arg at position 728.

Forward primer to MASP-3 SP domain:

GCGGCTAGCATGACTCTTCCAGAGTGTGGTCAGCCC,, SEQ ID NO:8

This primer comprises the NheI cleavage site, and the recombinantprotein will start with the Lys at position 433, which is preceded bythe usual Ala-Ser-Met-Thr sequence.

For CCP2-SP and CCP1-CCP2-SP construct the corresponding MASP-1 forwardprimers were used. In the PCR reaction human liver cDNA was used as atemplate. However, at a first attempt, no product was obtainedirrespective of the primer pair used.

Thereafter a “nested PCR” method was attempted. Two new primer pairswere designed corresponding to somewhat farther sequence sections.

Forward: CCTGTTCCATAGTGACAACTCGGGAGAGAA,, SEQ ID NO:9 Reverse:GGGAGGCAGGCCCCGAGGAAGTAAGTCAGC,, SEQ ID NO:10

With these primers apparently appropriate products were obtained. Thisproduct was used then as a template for the original primers. In thiscase products of appropriate size were obtained as demonstrated onagarose gels.

Construction of Recombinant Plasmids for Expression of the C1r Fragments

The cDNA fragments corresponding to the amino acids 309-705(CCP1-CCP2-SP), 376-705 (CCP2-SP) and 451-705 (SP) amino acids of humanC1r were amplified by PCR using the proof reading Pfu DNA polymeraseenzyme (Stratagene, La Jolla) and the full-length cDNA template (9). Forthe amplification procedure the following forward primers were obtainedfrom East Port Scientific (Budapest, Hungary):5′GCGAAGCTTGCCCCCAGCCCAAGACCCTA3′, SEQ ID NO:11,5′GCGAAGCTTGTGGGCAGCCCCGAAACCTG 3′, SEQ ID NO:12,5′GCGAAGCTTGTGGGAAGCCCGTGAACC3′, SEQ ID NO:13, in the case ofCCP1-CCP2-SP, CCP2-SP and SP, respectively. The reverse primer was5′GCGGTCGACTCAGTCCTCCTCCTCCATCT3′, SEQ ID NO:21, for each fragment. ThePCR products were digested with HindIII and SalI (cleavage sites arehighlighted) and were ligated into the HindIII/XhoI site of the pET-17bexpression vector (Novagen, Madison) in frame with the following Tagsequence 5′ATGTGCACCCAAGCT3′, SEQ ID NO:14. As a result of this therecombinant proteins contained four extra amino acids (Ser-Thr-Gln-Ala)at their N-terminus. The constructs were verified by DNA sequencing. Invitro mutagenesis experiments were carried out by means of theQuickChange site-directed mutagenesis kit (Stratagene, La Jolla, USA).The primer pairs (only the sense sequence is shown) wereGTGGAACAGAGGCAGCAGATAATCGGAGGGCAAAAAG, SEQ ID NO:15, for the Arg463Glnand GCCTGCCAGGGGGATGCTGGGGGCGTTTTTGCA, SEQ ID NO:16, for the Ser654Alamutations.

Expression, Renaturation, and Purification of the Recombinant MASPFragments

Expression—The expression plasmids were transformed into BL21(DE3) pLysShost strain, and the transformants were selected on Luria-Bertani mediumplates containing ampicillin and chloramphenicol. The expression wasconducted according to the manufacturer's instructions (pET systemmanual, 1997). After induction with isopropyl-D-thiogalactoside, thecells were collected in a 1/10 volume Tris-EDTA buffer and frozen at−20° C. The cells were then thawed and incubated for 30 min at roomtemperature in the presence of 0.5% Nonidet P-40. The viscous solutionwas sonicated to shear the DNA, and the inclusion bodies were collectedby centrifugation (12,000 g, 15 min, 4° C.). The supernatant wasdiscarded and the pellet was washed three times with Tris-EDTA buffer (1/10 of the culture volume).

Renaturation—The inclusion bodies were solubilized in 6 M GuHCl, 0.1 MTris-HCl (pH 8.3), 100 mM DTT at room temperature. The solutioncontained ˜10 mg/ml, or, in some cases 5 mg/ml protein. The solubilizedproteins were diluted 400-fold into the refolding buffers. The refoldingbuffers contained 50 mM Tris-HCl, 3 mM reduced glutathione, 1 mMoxidized glutathione, 5 mM EDTA, and 0.5 M arginine. The pH of thesolution was adjusted to pH 10.0 in the case of the MASP-2 SP fragmentand to pH 9.0 otherwise. Before the addition of the glutation thesolution was degassed and was cooled to 0° C. The renaturation processwas conducted at 6° C. overnight. The renatured protein solutions werethen dialyzed against 50 mM Tris-HCl (pH 9.0) and filtrated on a 0.45 μmnitrocellulose membrane.

Purification—The renatured proteins were purified on a Q-Sepharose-FastFlow column (Amersham Biosciences). The samples were loaded onto thecolumn and the elution was conducted with a linear NaCl gradient from 0to 400 mM. Fractions were analyzed by SDS-PAGE. The recombinant proteinswere further purified by cation exchange on Mono-S columns (AmershamBiosciences). The pH of the fractions containing the MASP-1 CCP1-CCP2-SPfragment and the MASP-2 SP fragment was adjusted to pH 5.0 and dialyzedagainst 50 mM AcOH (pH 5.0). The MASP-2 CCP-SP and MASP-2 CCP1-CCP-2-SPcontaining fractions were dialyzed against 20 mM Na-phosphate (pH 6.3).In each case a linear gradient of 0 to 600 mM NaCl was applied and thefractions containing the protein of interest were pooled. All proteinswere judged to be >90% pure by SDS-PAGE. Individual protein fragmentswere dialyzed against 20 mM Tris, 140 mM NaCl, pH 7.4, aliquoted, frozenin liquid nitrogen and kept at −20° C. The concentration of therecombinant proteins was determined by measuring absorbance at 280 nmusing the calculated absorption coefficients 18.7, 18.5, 19.1, and 14.9(1%, 1 cm) for the MASP-2 CCP1-CCP2-SP, CCP2-SP, SP, and MASP-1CCP1-CCP2-SP fragments, respectively. For calculation of the absorptioncoefficients we used the method of Gill et al. (ref.) taking disulfidebridges into account. The molecular masses calculated from the aminoacid sequences were 44,017, 35,722, 28,164, and 45,478 Da for the MASP-2CCP1-CCP2-SP, CCP2-SP, SP, and MASP-1 CCP1-CCP2-SP fragments,respectively.

Essentially the same way, MASP-3 fragments could be produced andrefolded.

Expression, Renaturation, and Purification of the Recombinant C1rFragments

Expression—The expression plasmids were transformed into the BL21(DE3)pLysS host strain and the transformants were selected on LB platescontaining ampicillin and chloramphenicol. The expression was carriedout according to the manufacturer's instructions [pET System Manual,Novagen Inc. (10)]. After induction with IPTG the cells were collectedin 1/10 volume TE buffer and freezed at −20° C. The cells were thenthawed and incubated for 30 min at room temperature in the presence of0.5% NP-40. The viscous solution was sonicated to shear the DNA, and theinclusion bodies were collected by centrifugation (12000 g, 15 min., 4°C.). The supernatant was discarded and the pellet was washed three timeswith TE buffer ( 1/10 of the culture volume).

Solubilization—The inclusion bodies were solubilized in 6 M GuHCl, 0.1 MTris-HCl pH 8.3, 100 mM DTT for 3 h at room temperature. The solutioncontained approximately 10 mg/ml protein. The solubilized proteins werediluted to 400-fold into the refolding buffers. The refolding bufferscontained 50 mM Tris-HC] pH 8.3, 3 mM GSH, 1 mM GSSG, 5 mM EDTA and 2 MGuHCl in the case of the CCP1-CCP2-SP fragment or 0.5 M arginine in thecase of the CCP-SP and SP fragments. The renaturation process wascarried out at 15° C. overnight. The renatured protein solutions werethen dialyzed against 50 mM Tris-HCl pH 7.4, 145 mM NaCl, filtrated on a0.45 μm nitrocellulose membrane and concentrated.

Purification—The renatured proteins were purified on Q-Sepharose-FastFlow column (Pharmacia, Uppsala, Sweden). The samples were dialyzedagainst and the column was equilibrated in a buffer containing 20 mMNaCl and 20 mM Tris-HCl pH 8.3 for the CCP1-CCP2-SP and CCP2-SP or pH7.4 for the SP fragments. The samples were loaded onto the column andthe elution was carried out with a linear NaCl gradient from 20 mM to400 mM, Fractions were identified by SDS-PAGE. The recombinant proteinswere further purified by gel filtration using a Superose-12 FPLC column(Pharmacia, Uppsala, Sweden) in 50 mM NaCl, 20 mM Tris-HCl pH 8.3 forCCP1-CCP2-SP and CCP2-SP or pH 7.4 for SP. The concentration of therecombinant proteins was determined by absorbance at 280 rim using theabsorption coefficients 15.2, 15.8, 15.4 (1%, 1 cm) for theCCP1-CCP2-SP, CCP2-SP, and SP fragments, respectively. For calculationof absorption coefficients we used the method of Gill et al. (11) takingdisulfide bridges into account. The molecular masses calculated from theamino-acid sequences were 45532 Da, 37670 Da, and 28976 Da forCCP1-CCP2-SP, CCP2-SP, and SP fragments, respectively.

C1s fragments could be produced essentially the same way.

Purification of C2 Factor I, and Factor H

Human C2 was prepared by a recent immunoaffinity method (Laich and Sim,2001). It was dialyzed against 20 mM HEPES, 140 mM NaCl, pH 7.4, andfrozen in liquid nitrogen. Extinction coefficient (280 nm, 1%, 1 cm) wasestimated from its amino acid sequence to be 9.4 for the 102 kDaprotein.

Factors H and I were purified by the method of Sim et al. (1993).Extinction coefficients, ε(280 nm, 1%, 1 cm), of 12.4 and 14.3, andmolecular weights of 150 kDa and 88 kDa were used for Factors H and I,respectively (Pangburn and Muller-Eberhard, 1983).

Purification of C3 and C4

Human C4 was purified according to the method of Dodds (1993) withmodifications. Briefly, fresh citrated plasma (15 ml) was made 1 mMPefabloc-SC and 10 μg/ml soy bean trypsin inhibitor type I-S (SBTI-IS)[Sigma], then precipitated with 50 mM barium chloride for 1 h on ice.The supernatant was recovered by centrifugation and made 1 mMPefabloc-SC and 5 mM EDTA. The plasma was then precipitated with 5%(w/v) polyethylene glycol (PEG 3350 molecular weight) [Sigma] for 30 minon ice, by adding 15% (w/v) PEG in Buffer A (20 mM Tris, 50 mMε-aminocaproic acid (εACA), 5 mM EDTA, 0.02% NaN₃, pH 7.4), and thesupernatant recovered as before. A column of Q-Sepharose Fast Flow(16/10) [Amersham] was equilibrated in 95% Buffer A, 5% Buffer B (20 mMTris, 50 mM εACA, 5 mM EDTA, 0.02% NaN₃, 1 M NaCl, pH 7.4), and thesupernatant was applied to the column. Proteins were eluted with alinear salt gradient. C3 eluted from the Q-Sepharose column in thesecond peak, in the 200-250 mM NaCl range, and fractions containing C3,as observed by SDS-PAGE, were pooled. The C3 pool was diluted with halfa volume of water and made 1 mM Pefabloc-SC. A column of Mono-Q (HR 5/5)[Amersham] was equilibrated with 90% Buffer A, 10% Buffer B, and thedilute C3 pool was loaded and eluted with a linear gradient. C3 elutedas a single peak and was judged >90% pure by SDS-PAGE. It was alsoestimated to be >95% ‘live’ (i.e., its thiol ester was intact) by thepresence of autocatalytic cleavage fragments of C3 α-chain (Sim and Sim,1981); and the lack of cleavage by Factor I in the presence of Factor H.

C4 eluted from the Q-Sepharose column in the final peak, and fractionscontaining C4, as observed by SDS-PAGE, were pooled. The C4 pool wasdiluted with half a volume of distilled water, loaded onto a Mono-Q FPLCcolumn and eluted in a linear salt gradient. C4 was determined tobe >80% pure by SDS-PAGE.

Live C3 and C4 were dialyzed against 20 mM HEPES, 140 mM NaCl, pH 7.4.Live C3 was used within three days, C4 was frozen in liquid nitrogen in200 μl aliquots and kept at −80° C. for storage. Aliquots were thawedonly once and then used within five days of thawing to ensure theproteins were active. Extinction coefficients, ε(280 nm, 1%, 1 cm), of9.7 and 8.3, and molecular weights of 185 kDa and 205 kDa were used forC3 and C4, respectively (Tack and Prahl, 1976; Nagasawa and Stroud,1977).

Purification of C1-inh and Alpha-2-Macroglobulin

C1-inh was purified from human serum using protocols by Sim and Reboul(1981) and Pilatte et al (1989). Extinction coefficient, ε(280 nm, 1%, 1cm), of 3.6 and molecular weight of 71.1 kDa were used (Aulak et al.,1993).

Human alpha-2-macroglobulin (α₂M) was isolated first as a by-product ofthe method for C3 and C4 purification (Dodds, 1993), then furtherpurified according to Salvesen and Enghild (1993). Extinctioncoefficient, ε(280 nm, 1%, 1 cm), of 9.0 and molecular weight of 720 kDawere used (Salvesen and Enghild, 1993).

Amidolysis of C3 The intact thiol ester bond in ‘live’ C3 was cleavedusing ammonium salt as the nucleophile to obtain ‘dead’ C3(NH₃),according to Soames and Sim (1997) with modifications. C3 was incubatedwith a final concentration of 0.2 M ammonium hydrogen carbonate for 90min at 37° C., making sure that the final pH of the reaction was above8.0. At the end of the incubation, the reaction was collected with abrief centrifugation (5 min, 8000 g, RT) and dialyzed against 20 mMHEPES, 140 mM NaCl, pH 7.4.

N-terminal sequencing After SDS-PAGE and blotting to polyvinylidenedifluoride membrane, the N-terminal amino acid sequences of therecombinant proteins were determined by a pulsed-liquid phase proteinsequencer ABI 471A.

Oligopeptide Substrate Library

The specificities of the MASP-1 and MASP-2 CCP1-CCP2-SP fragments weretested on a competing oligopeptide library applying the method describedbefore (Antal, 2001).

Functional Studies with MASP Fragments

C2, C3, C3(NH₃), C4 cleavage—Serial dilutions of MASP-1 and MASP-2fragments ranging between concentrations of 1 μM and 10 pM wereincubated at 37° C. in 20 mM HEPES, 140 mM NaCl, pH 7.4 with C2, C3,C3(NH₃) or C4 at typical concentrations of 0.5-1 μM. The cleavage wasfollowed by SDS-PAGE under reducing conditions. Desirable concentrationsof the MASP fragments for further detailed kinetic analysis were chosento be where half of the protein substrates were hydrolyzed during thefirst 20 minutes of incubation. The protein substrates C2, C3, C3(NH₃)or C4 were then incubated with the selected concentrations of the MASPfragments at 37° C. in 20 mM HEPES, 140 mM NaCl, pH 7.4. Typically 11-13samples were taken at varying time periods but always within 50 minutesfrom the beginning of the reaction. Cleavages rates were quantified bymeasuring the diminution of the cleaved chain visualized by Coomassiestained SDS-PAGs using a GEL DOC 1000 instrument and Molecular AnalystSoftware for densitometric calculations (Bio-Rad, Hercules, Calif.,USA). The reactions were assumed to be of the Michaelis-Menten type. Inthe case of C3 and C3(NH₃) cleavage a further reasonable assumption ofK_(M)>>[substrate] was made. The constants K_(M), k_(cat) andK_(M)/k_(cat) were estimated by unbiased non-linear regression methodsregressing the data on the following equations:t=([S₀]−[S]+K_(M)ln([S₀]/[S]))/(k_(cat)[E₀]) or in the case of C3 andC3(NH₃) cleavage on [S]=[S₀]exp(−t[E₀]k_(cat)/K_(M)).

Reactivity toward C1-inhibitor and alpha-2-macroglobulin—Inhibition ofthe enzymatic activities of the MASP fragments by C1-inhibitor andalpha-2-macroglobulin were tested as follows. MASP-1 and MASP-2fragments at concentrations selected in the C2, C3, C3(NH₃) and C4cleavage tests were incubated at 37° C. with excess molar ratios ofeither C1-inhibitor, alpha-2-macroglobulin, or buffer in 20 mM HEPES,140 mM NaCl, pH 7.4 for 40 minutes. Complement substrates C2, C3,C3(NH₃) or C4 were added at typical concentrations of 0.5-1 μM and thesamples were further incubated for 20 minutes. The cleaved and uncleavedchains were visualized by reducing SDS-PAGE and the degree of inhibitionwas compared.

To assess the role of both C1-inhibitor and alpha-2-macroglobulin in theregulation of MASP-1 and MASP-2 the following experiment was devised.Each of the ¹²⁵I-labeled MASP fragments was incubated with eitherC1-inhibitor or alpha-2-macroglobulin or both at physiological relativeconcentrations ([MASP-1]: [C1-inh]: [α₂M]=1:30:60; [MASP-2]:[C1-inh]:[α₂M]=1:90:180) at concentrations [MASP-1 CCP1-CCP2-SP]=9 nM,[MASP-2 fragments]=3, nM at 37° C. in 20 mM HEPES, 140 mM NaCl, pH 7.4for 90 minutes. The samples were further incubated at 37° C. for 30minutes, run on reducing SDS-PAGE and visualized by means ofautoradiography. The following serum concentrations were used whencalculating the relative concentrations: [MASP-1]=65 nM (Terai et al.,1997), [MASP-2]=22 nM (Hajela, 2002), [C1-inh]=2 μM (Terai et al., 1997)and [alpha-₂M]=4 μM.

To assess the rate of SDS stable complex formation between the MASP-1and MASP-2 CCP1-CCP2-SP fragments and C1-inhibitor, the ¹²⁵I-labelledMASP fragments at concentrations ranging between 30-100 nM wereincubated with 100-350 nM C1-inhibitor at 37° C. in 20 mM HEPES, 140 mMNaCl, pH 7.4 for 40 minutes. Typically 11-13 samples were taken atvarying time periods. The reactions were stopped by reducing samplebuffer containing 0.125 M Tris, 4.8% SDS, 8 M urea 20% glycerol, 720 mMmercaptoethanol, 0.02% bromophenol blue, pH6.8. The samples were furtherincubated at 37° C. for 30 minutes and run on reducing SDS-PAGE.Cleavages rates were visualized by means of autoradiography and thediminution of the reacted MASP chain was quantified using a GEL DOC 1000instrument and Molecular Analyst Software for densitometric calculations(Bio-Rad, Hercules, Calif., USA). The kinetic constants were calculatedassuming irreversible inhibition proceeded by reversibleenzyme-inhibitor complex formation. Using steady-state approximation andneglecting the starting enzyme concentration compared to the startinginhibitor concentration ([I₀]>>[E₀]) the data was regressed on thefollowing equation using non-linear regression methods:[E]=[E₀]exp(−k_(obs)t); k_(obs)=k₂[I₀]/(K_(i)+[I₀]), where k_(obs) isthe observed pseudo-first order rate of reaction, k₂ is the rate ofirreversible enzyme-inhibitor complex formation and K_(i) is theinhibitory constant.

Radio Iodination and Autoradiography of MASP-Fragments

Radioiodination—The MASP-1 CCP1-CCP2-SP, MASP-2 CCP1-CCP2-SP, CCP2-SPand SP fragments were labeled with ¹²⁵I using iodogen(1,3,4,6-tetrachloro-3α,6α-diphenylglycoluril) [Sigma] as the oxidisingagent (Raker and Speck, 1978). Eppendorf tubes (1.5 ml) were coated with200 μl iodogen and excess iodogen was washed off with 0.5 ml 20 mMHEPES, 140 mM NaCl, 5 mM CaCl₂, pH 7.4 (HBS). Protein samples weretransferred to the iodogen-coated tube and incubated with 10 μl Na-¹²⁵(1 mCi) [Amersham International, Aylesbury, UK] for 10-20 min on ice.Free 125-iodide was removed by desalting on a PD-10 Sephadex G-25 gelfiltration column [Amersham] which was presaturated with 0.1% (v/v)Emulphogene BC720 [Sigma] in HBS. Radiolabelled protein fractions werepooled and stored at 4° C. Specific activity of a protein was determinedby measuring on a Mini-Assay type 6-20 manual y counter [MiniInstruments, Burnham-on-Crouch, Essex, UK].

Autoradiography—Dried SDS-PAGE gels of ¹²⁵I-labelled samples wereexposed to X-ray film [Fuji RX, Fuji Photo Film UK Ltd, London] at −70°C. in autoradiography cassettes with intensifying screens. Films weredeveloped using an X-Ograph imaging system Compact X4 [X-Ograph imagingsystem, Malmesbury, UK].

Enzymatic Assays with the C1r Fragments

Esterolytic activity—The rates of hydrolysis were measured on theZ-Lys-S-Bzl, and Z-Gly-Arg-S-Bzl thioesters. The release rate of HS-Bzlwas measured through its reaction with 4,4′-dithiodipyridine (12), andwas followed with a Jasco V-550 spectrophotometer at a wavelength of 324nm. Assays were carried out following the method of McRae et al. (13) at30° C. in 20 mM Tris buffer at pH 7.5 containing 145 mM NaCl.k_(cat)/K_(M) values were directly determined from the catalytic rate atlow substrate concentrations (10-30 μM).

C1s cleavage—Proenzyme C1s was expressed in baculovirus expressionsystem using High Five insect cell culture. Functionally pure (80%) C1sproenzyme was obtained by purifying the cell culture supernatant on aDEAE Sepharose FF column (Amersham Pharmacia Biotech) as described in(14). C1s preserved its proenzyme state during the purification andstorage. Proenzyme C1s cleavage ability of the C1r fragments were testedby means of the esterolytic activity of the generated active C1smolecules on the Z-Lys-S-Bzl thioester substrate. An enzyme/C1s molarratio of 1:50 was used for most of the experiments. 10-15 ml C1sproenzyme solution with a protein concentration of ˜0.1 μM in 20 mMTris, 145 mM NaCl buffer pH 7.4 was thermostated at 30° C. The C1r SP,CCP2-SP and CCP1-CCP2-SP fragments were added to it at a finalconcentration of 1−3×10⁻⁹ M. At 1 min intervals 1 ml of the mixture waswithdrawn and the esterolytic activity was measured by the addition ofthe Z-Lys-S-Bzl substrate at a final concentration of 100 μM. Themaximal specific activity value of totally activated C1s of 182 s⁻¹ at100 μM Z-Lys-S-Bzl concentration was used for the calculation of theactual concentration of C1s. k_(cat)/K_(M) values were calculated bylinear fitting for the first 5-8 points where the amount of the cleaved,active C1s was less than 10% of the total proenzyme concentration.

C1r autoactivation experiments—The S654A mutant proenzyme SP, CCP2-SP,and CCP1-CCP2-SP fragments were used as substrates to investigate theautoactivation ability of the wild-type active C1r fragments.Measurements were carried out in 20 mM Tris, 145 mM NaCl buffer at pH8.3. Reaction was started by the addition of the active enzyme to theS654A mutant proenzyme solution thermostated at 37° C. An enzyme:zymogenratio of 1:10-1:50 was used. 10 μl aliquots were removed at 10-15 timepoints in the range of 0.5 min-1 h and added to 10 μl 5% SDS samplebuffer (15) containing 5% mercaptoethanol and were immediately incubatedfor 3 min at 100° C. The cleavage at the activation site of theproenzyme molecules was followed by reducing SDS-PAGE. The acrylamidegels were stained with Coomassie brilliant blue. The concentration ofthe uncleaved proenzyme vs. time was calculated from the density of itsbands recorded by a BioRad GelDoc2000 imaging system. After curvefitting and derivation in Origin 5.0 data analysing software (MicroCalInc.) the cleavage rate vs. proenzyme concentration was calculated andproved to be linear in the concentration range used, thereforek_(cat)/K_(M) values could be obtained from the slope of the curve.

Differential Scanning Calorimetry (DSC)

Calorimetric measurements were performed on a VP-DSC (Microcal Inc,Northampton, Mass.) differential scanning calorimeter. Denaturationcurves were recorded between 10 and 80° C. at a pressure of 2.5 atm,using a scanning rate of 1° C./min. The protein concentration was set to0.1 mg/ml. Samples were dialyzed against 20 mM Tris pH 8.3 145 mM NaCl,and the dialysis buffer was used as a reference. Heat capacities werecalculated as outlined by Privalov (16).

Zymography

Protocols for gelatine zymography are described e.g. in (Kleiner at al.1994) and in (Liota, 1990) which are incorporated herein by reference.

2. Results Obtained with the MASP Fragments

Expression, Purification and Characterization of the Recombinant MASPFragments

The catalytic region of MASP-1 and MASP-2, consisting of the twocomplement control protein modules and the serine protease domain(CCP1-CCP2-SP) (FIG. 3.) was expressed in E. coli BL-21 cells using thepET-17b expression vector. In the case of MASP-2, truncated fragments ofthe catalytic region (the serine protease domain with one CCP module andthe serine protease domain alone) (FIG. 3) were also expressed in thesame expression system. Since the recombinant proteins accumulated asinclusion bodies inside the bacterial cells, renaturation procedureswere needed to restore the native, folded structure (see Materials andMethods). The renatured recombinant proteins were purified by ionexchange chromatography on Q-Sepharose Fast Flow and Mono-S columns. Therenaturation and the purification procedures were followed by SDS-PAGEand the purified proteins were subjected to N-terminal sequencing.Before renaturation the inclusion body proteins yielded single bands onthe reducing SDS-PAGE corresponding to the zymogen form of the enzymes.After renaturation the MASP-1 CCP1-CCP2-SP fragment was fully activatedand yielded two major bands under reducing conditions, whichcorresponded to the two-chain-structure obtained by the cleavage of theactivation site Arg⁴⁴⁸-Ile⁴⁴⁹ bond, as determined by sequencing. TheMASP-1 CCP1-CCP2-SP fragment co-purified with a minor contaminant, whichmigrated on SDS-PAGE as a band of 24 kDa under reducing and at 39 kDaunder non-reducing conditions. N-terminal sequencing revealed that thisfragment is a degraded form of the MASP-1 catalytic region lacking a 6kDa fragment from its serine protease domain. The cleavage occurred atthe Arg⁵⁰⁴-Asp⁵⁰⁵ bond removing the histidine from the catalytic triadand thus causing the loss of its enzymatic activity. The MASP-2fragments migrated as single chain structures under reducing conditionsthroughout the renaturation procedure, however, they became activatedduring the purification process. Sequencing analysis confirmed theactivation of the MASP-2 fragments to occur through the cleavage of theArg⁴⁴⁴-Ile⁴⁴⁵ bond and further modifications were observed in the caseof the MASP-2 CCP1-CCP2-SP fragment, which displayed Ile²⁹¹ at itsN-terminus. This indicated that the bond between Lys²⁹⁰-Ile²⁹¹ wascleaved and a heptapeptide (Ala-Ser-Met-Thr-Gly-Trp-Lys, SEQ ID NO:17,)was completely removed. This cleavage, however, does not compromise theintegrity of the MASP-2 CCP1CCP2-SP construct, since for technicalreasons the original N-terminus contained three extra amino acids(Ala-Ser-Met of the T7-Tag) and the CUB2-CCP1 junction is atHis²⁹²-Tyr²⁹³. The yield of proteins produced in the form of inclusionbodies in the cell culture was judged to be between 10-40 mg/l prior torenaturation. The overall yield pertaining to the amount of purifiedproteins recovered from one liter of cell culture ranged between 0.1-0.5mg/l. A shift in pH to below pH 8.5 (8.3) resulted in at least a 3 timesdecrease in yield, in some cases (e.g. MASP-2 SP fragment) more or noyield at all.

Mutant Constructs

The expression and purification system of the invention proved to bevery useful to prepare mutant MASP-fragments. Up to the present fourconstructs encoding mutant CCP1-CCP2-SP fragments are prepared. In theMASP-1 and MASP-2 CCP1-CCP2-SP fragments the activation siteArg⁴⁴⁸-Ile⁴⁴⁹ and Arg⁴⁴⁴-Ile⁴⁴⁵ bonds were mutated to Gln⁴⁴⁸-Ile⁴⁴⁹(R448Q) and Gln⁴⁴⁴-Ile⁴⁴⁵ (R444Q) respectively. The latter mutantfragment was expressed and purified to homogeneity using the method asdescribed above. About 1 mg of protein was obtained, which was notautoactivated but could be activated by lysosyme.

In the other two mutant constructs the codons of the active site serineswere changed to codons of alanine: MASP-1 S(646)A and MASP-2 S(633)Amutants.

Proteolytic Activity of MASP-1 on Complement Components

The proteolytic activity of the MASP-1 catalytic fragment (CCP1-CCP2-SP)was investigated on protein substrates that are involved in theformation of the C3 and CS convertase enzyme complexes of the classicaland lectin pathways (i.e. on C2, C4, and C3). To resolve the controversyabout the C3 cleaving ability of MASP-1 both ‘live’ and ‘dead’ C3 wasused in the assays. ‘Live’ C3 (i.e. C3 with an intact thiol ester bond)was isolated from fresh human serum and used within three days in themeasurements. It was rigorously tested before the proteolyticmeasurements by co-treating it with Factor I and Factor H and wasfound >95% ‘live’. To test the proteolytic activity of MASP-1 on ‘dead’C3 (i.e. on C3 with a cleaved thiol ester bond), a batch of the C3preparation was inactivated using ammonia as the small nucleophileleading to C3(NH₃) formation. The characteristic k_(cat)/K_(M) valuesshow that the MASP-1 CCP1-CCP2-SP fragment cleaves ‘dead’ C3 with a lowbut significant efficiency, whereas its proteolytic action on ‘live’ C3is about 20 fold less (Table 1, FIG. 5.B). Nevertheless, it is importantto note that in control experiments using the C1r CCP1-CCP2-SP fragmentexpressed in the same expression system as the MASP fragments (Kardos etal. 2001) we observed no cleavages of either ‘live’ or ‘dead’ C3. Ourexperiments demonstrated that complement component C4, similarly to‘live’ C3, is basically resistant against the proteolytic activity ofMASP-1 (Table 2, FIG. 5.C). C2, however, was digested by our MASP-1fragment at a moderate rate (Table 2, FIG. 5.A). All cleavages could beinhibited by preincubating MASP-1 CCP1-CCP2-SP with a molar excess (1-3fold) of either C1-inhibitor or alpha-2-macroglobulin (FIG. 6).

Proteolytic Activity of MASP-2 on Complement Components

The proteolytic activity of the MASP-2 CCP1-CCP2-SP fragment on C3 wasvery similar to that of the MASP-1 fragment. While MASP-2 CCP1-CCP2-SPhad only a hardly detectable marginal activity on ‘live’ C3, itexhibited a low but significant enzymatic activity on ‘dead’ C3 (Table1, FIG. 5.B). In contrast to the MASP-1 fragment the CCP1-CCP2-SP,CCP2-SP and SP fragments of MASP-2 cleaved C2 and C4 very efficiently(Table 2, FIG. 5.A, C). The comparison of the three MASP-2 fragmentsshows that the serine protease domain of MASP-2 on its own can cleave C2with high efficiency. The addition of the CCP modules to the SP domainsomewhat decreases the proteolytic power in C2 cleavage. When C4 is usedas a substrate, the MASP-2 fragments show a different picture. Althoughthe serine protease domain on its own is capable of cleaving C4, thepresence of the CCP2 domain significantly increases the efficiency ofthe catalysis (a 44-fold increase in the k_(cat)/K_(M) value). TheCCP1-CCP2-SP fragment of MASP-2 is also very efficient in cleaving C4,although its proteolytic power is less than that of the CCP2-SPfragment. All cleavages could be abolished by preincubating the MASP-2fragments with a molar excess (1-3 fold) of C1-inhibitor (FIG. 5) orwith a large molar excess (40 fold) of alpha-2-macroglobulin (FIG. 6).

Proteolytic Activities of the Catalytic Fragments of MASP-1 and MASP-2on Oligopeptide Substrates

To further investigate the range of substrate specificity and relativespecific activities of MASP-1 and MASP-2 we applied a competingoligopeptide substrate library. (Antal, 2001). This library contains amixture of seven oligopeptides (each 13 amino acids in length) withdifferent cleavage sites (P1) for both trypsin and chymotrypsin-likeenzymes (His-Ala-Ala-Pro-Xxx-Ser-Ala-Asp-Ile-Gln-Ile-Asp-Ile, SEQ IDNO:18, where Xxx could be Lys, Arg, Tyr, Leu, Phe, Trp or Pro). Theindividual peptide substrates compete for the protease underinvestigation, in our cases for either the MASP-1 CCP1-CCP2-SP or theMASP-2 CCP1-CCP2-SP fragment. The results show that both enzymes cleaveoligopeptides that contain either Arg or Lys at their P1 positions. TheMASP-1 catalytic fragment (CCP1-CCP2-SP) exerted a substantially largeractivity on the Arg oligopeptide substrate than the MASP-2 fragment(FIG. 7). Comparing the relative specific activities of the twofragments on the P1 Arg oligopeptide under the same conditions, at 15min MASP-1 showed almost 90% digestion, whereas MASP-2 cleaved less than20% of the substrate. This result substantiates that MASP-1 is a potentprotease with significant catalytic strength. Another essential outcomeof the competing oligopeptide substrate library experiments is thatMASP-1 possesses extreme Arg selectivity at the P1 site of itssubstrate. This behavior strongly resembles that of thrombin, whichexhibited similar Arg selectivity in the same competing oligopeptidelibrary system (Antal, 2001). MASP-2 also preferred the Arg substrate tothe Lys one, but the degree of preference was much less than that ofMASP-1. A very similar specificity profile was obtained for trypsin inthis oligopeptide library system.

Zymography provided an additional evidence that the substratespecificity of MASP-1 is significantly broader than that of MASP-2.

The Reaction of the MASP-1 and MASP-2 Fragments with C1-Inhibitor andAlpha-2-Macroglobulin

As mentioned in the previous chapters, C1-inhibitor completely abolishedthe proteolytic activity of both MASP-1 and MASP-2 on all substrates. Amarginal molar excess of C1-inhibitor over the enzyme concentrationproved to be sufficient for full inhibition. Another inhibitor,alpha-2-macroglobulin (alpha-₂M), also inhibited the reactions, butexhibited lower efficiency toward MASP-2. The inefficient C3 cleavagescould all be blocked by a small 1-3 fold molar excess over MASP-1CCP1-CCP2-SP or MASP-2 CCP1-CCP2-SP, whereas only a 40-fold molar excessover the MASP-2 fragments was capable of significantly hindering C2 andC4 cleavage. The moderate cleavage of C2 by the MASP-1 CCP1-CCP2-SPfragment could be completely blocked by a small 1-3 fold molar excess ofalpha-2-macroglobulin. To confirm the inhibition of the MASPs by α2M andto judge the relative rates of inhibition by a2M and C1-inhibitor theMASP fragments were labeled with ¹²⁵I. The radiolabeled MASPs were thenmixed with C1-inhibitor, α2M or both, in serum-like relativeconcentrations and the enzyme-inhibitor complexes were analyzed bySDS-PAGE followed by autoradiography (FIG. 6). Both C1-inhibitor and α2Mformed SDS-PAGE stable complexes with all MASP fragments. In the case ofthe MASP-2 fragments C1-inhibitor proved to be the primary inhibitor, asit reacted faster with the MASP-2 fragments than the α2M, whereas in thecase of MASP-1 alpha-₂M is the more probable physiological inhibitor.The observed pseudo first order rates of reaction (k_(obs)) with C1-inhibitor were 5-fold less for the MASP-1 CCP1-CCP2-SP than for theMASP-2 CCP1-CCP2-SP fragment. The K_(i) values for the C1-inhibitor andMASP-2 CCP1-CCP2-SP reaction were in the nanomolar range (Table 3).

3. Results Obtained with the C1r Fragments

Expression and Renaturation of Recombinant Proteins

Three cDNA fragments from the catalytic region of C1r (FIG. 9) have beencloned into a modified pET-17b vector in fusion with the(Met)-Ser-Thr-Gln-Ala sequence. Each insert begins with a Cys (Cys³⁰⁹,Cys³⁷⁶, Cys⁴⁵¹ in the case of CCP1-CCP2-SP (γB), CCP2-SP and SP,respectively) at the N-terminus and ends with Asp⁷⁰⁵ at the C-terminus.The mature proteins have an N-terminal sequence Ser-Thr-Gln-Ala-(Cys) .. . as verified by protein sequencing. In order to preventautoactivation, stabilized mutant constructs were also expressed. In oneseries we introduced the Arg463Gln mutation into the cDNAs, while inanother series we changed the active site Ser654 to Ala for all thethree fragments. The expression plasmids were transformed into the E.coli BL21(DE3) pLysS strain and the recombinant protein expression wasinduced by adding IPTG. After induction the cells were lysed and thesoluble and insoluble fractions were separated by centrifugation andanalyzed on SDS-PAGE (data not shown). In the soluble fraction we couldnot detect recombinant proteins using Coomasie blue staining, whereasthe pellet contained almost exclusively the recombinant C1r fragments(purity approx. 80%). Since the recombinant proteins were present asinclusion bodies, renaturation procedures were needed to generate thenative, folded structure. The inclusion bodies were solubilized in 6 Mguanidine-HCl solution, which contained 100 mM DTT to reduce all thedisulfide bridges. The solubilized recombinant proteins (˜10 mg/ml) werethen diluted 400-fold (final concentration ˜25 μg/ml) using differentrefolding buffers and incubated at 15° C. overnight. Many differentrefolding solutions containing various additives and different oxidoshuffling reagents were tested for the three fragments and the best oneswere selected for large scale renaturation (17, 18). We found thehighest renaturation yield using 2 M guanidine-HCl in the case ofCCP1-CCP2-SP fragment and 0.5 M L-arginine in the case of CCP2-SP and SPfragments. The optimal oxido shuffling system was the mixture of reducedand oxidized glutathione in a ratio of 3 mM GSH/1 mM GSSG at pH 8.3 inall experiments. The efficiency of the folding process could beestimated by reducing SDS-PAGE, since native, functionally active C1rcan cleave itself into two chains (γ 18 kDa and B 30 kDa chains in thecase of the CCP1-CCP2-SP fragment). Since the denatured recombinantproteins have single-chain structure in the inclusion bodies, theappearance of two chains on the reducing gel is a good indicator ofautoactivation and hence of successful renaturation of the wild typefragments (FIG. 10A). Edman degradation of the large (30 kDa) chainsyielded the Ile-Ile-Gly-Gly-Gln sequence in all cases, indicating thatthe correct autolytic cleavage at the Arg⁴⁶³-Ile⁴⁶⁴ bond between the γand B chains had occurred during the activation process. The efficiencyof the renaturation was about 10-20%, allowing us to obtain enoughmaterial for all subsequent physico-chemical and functional studies.After the renaturation process the aggregated material was removed byfiltration on a 0.45 μm nitrocellulose membrane, and the refoldedrecombinant proteins were purified by anionexchange and gel-filtrationchromatography as described in the Experimental Procedures. On theQ-Sepharose Fast Flow column most of the contaminants did not bind tothe resin at low ionic strength (20 mM NaCl and 20 mM Tris-HCl) andcould be removed by washing the column with the low salt buffer. Thecorrectly folded, native recombinant fragments eluted as single peaksdetected at 280 nm during the ascending salt gradient. There was nodifference between the elution volume of the wild-type (activated) andthe R463Q or S654A mutant (zymogen) fragments. The recombinant fragmentswere essentially pure after the ion-exchange chromatography, however toremove the traces contaminants we performed a gel filtrationchromatography on a Superose-12 FPLC column. After this step the proteinsolutions were concentrated and the concentrations of the recombinantproteins were measured from the absorbance at 280 nm. The final yieldsfor the CCP1-CCP2-SP, CCP2-SP and SP fragments were 2, 5, and 2 mg/l ofculture, respectively. Both the wild-type and the zymogen mutantfragments yielded a single band on non-reducing SDS-PAGE analysis,although with different apparent molecular weights (˜45 kDa and ˜39 kDafor wild-type CCP1-CCP2-SP and zymogen CCP1-CCP2-SP, respectively, ˜38kDa and ˜34 kDa for wild-type and zymogen CCP2-SP, and ˜31 kDa and ˜27kDa for the wild-type and zymogen SP fragment). On the reducing gelhowever the wild-type fragments exhibited the activated two-chain forms(˜30 kDa for the B chain and ˜18 kDa for the γ chain), whereas thezymogen mutants retained a single-chain structure. In order to prove thecorrect folding of the zymogen mutants, the renatured proteins wereconverted into the two-chain form. The Arg463Gln mutants could bespecifically cleaved at the Gln⁴⁶³-Ile⁴⁶⁴ and activated by thermolysin(FIG. 10B). After thermolysin treatment all the three R463Q fragmentsshowed proteolytic and esterolytic activity similar to that of thewild-type autoactivated proteases (Table 5). The Ser654Ala mutantscannot autoactivate themselves, but wild-type C1r fragments could cleavethem at Arg⁴⁶³-Ile⁴⁶⁴ activation site, as verified by proteinsequencing.

Physico-Chemical Characterization

Gel permeation chromatography—relative molecular weight—In order todetermine the relative molecular weight and investigate the dimerizationproperties of the expressed and renaturated C1r fragments, the relativemolecular weights of the three C1r fragments vs. pH were analyzed by gelpermeation chromatography. The molecular weights of the three C1rfragments were determined relative to the standards at every pH. (FIG.11).

The SP and the CCP2-SP fragments showed molecular weights of 28-30 kDaand 36-38 kDa, respectively. These values were independent of pH andwere in accord with the molecular weights determined from SDS-PAGEanalysis. The relative molecular weight of the CCP1-CCP2-SP fragment wasfound to be approximately 90 kDa at neutral or alkaline pH. Below pH 6.0it showed a sigmoidal shaped decrease to 44-47 kDa, which is themolecular weight of the monomer CCP1-CCP2-SP, and is in accord with theSDS-PAGE.

Differential Scanning Calorimetry—DSC measurements were performed on onehand to check the native structure of the fragments and on the otherhand to investigate the role of the individual domains in theconformational stability of the catalytic region of C1r.

The SP fragment showed a sharp, cooperative melting transition at arelatively low temperature (47.5° C.), indicating a compact, stablestructure of the molecule (FIG. 12).

Fragment CCP2-SP showed a cooperative unfolding curve with a meltingpoint at 55.4° C. The larger calorimetric enthalpy change and thesignificantly higher melting temperature compared to that of SP indicatethat the CCP2 module establishes tight interactions with the SP domainand significantly improves its stability.

The CCP1-CCP2-SP fragment showed an unfolding transition at 57.5° C.,which is in good agreement with that of the equivalent CCP1-CCP2-SPproduced in baculovirus expression system (58.3° C.) (19). These dataprove that renatured CCP1-CCP2-SP is in a native form and that itsconformational stability is similar to that of a CCP1-CCP2-SP with asomewhat larger N-terminal and with carbohydrate side-chains. Thestability of CCP1-CCP2-SP is somewhat higher than that of CCP2-SP. Thepresence of the CCP1 module and the dimerization of the CCP1-CCP2-SPfragment exert less effect on the stability of the protein as comparedto the significant stabilizing effect of the CCP2 module in theinteraction with SP domain in the CCP2-SP construct.

Functional Characterization of the Recombinant Proteins

Esterolytic activity on synthetic substrates—The values of the catalyticefficiency (k_(cat)/K_(M)) for the reaction of the C1r fragments withthe Z-Lys-S-Bzl, and Z-Gly-Arg-S-Bzl thioesters are presented in Table5. Z-Lys-S-Bzl is not a “good” substrate for C1r, but its spontaneoushydrolysis rate is very low, therefore measurements of low catalyticactivity was possible. Z-Gly-Arg-S-Bzl, a more sensitive thioestersubstrate of C1r was hydrolyzed at high rate by the fragments. The threeC1r fragments showed similar esterolytic activities on the thioestersubstrates indicating similar active site conformations in the SP,CCP2-SP and CCP1-CCP2-SP fragments. The CCP2-SP fragment, however provedto be slightly more potent as compared to the others. The observedk_(cat)/K_(M) values on the Z-Gly-Arg-S-Bzl substrate are four timeshigher than those described previously for the entire C1r moleculeisolated from human serum (13),

The catalytic efficiency of C1s cleavage by the recombinant C1rfragments—The ability of the C1r fragments to cleave proenzyme C1s wastested through the esterolytic activity of the activated C1s moleculeson the Z-Lys-S-Bzl thioester substrate. Although C1r also cleaves thissubstrate (see Table 5), its catalytic efficiency is about two orders ofmagnitude less than that of C1s. An enzyme-substrate ratio of 1:50assures that the activity of C1r on the Z-Lys-S-Bzl was negligiblecompared to that of C1s. The proenzyme C1s concentration (0.04-0.1 μM)was orders of magnitude below the K_(M) value and this allowed directcalculation of the k_(cat)/K_(M) values from the linear part of the C1sactivation curve. Kinetic analysis of the activation of C1s byCCP1-CCP2-SP is shown on FIG. 13. The slope of the line is proportionalto the catalytic efficiency. The results with the three C1r fragmentsare summarized in Table 6. All the three fragments efficiently cleavedproenzyme C1s. The CCP2-SP fragments exhibited an exceptionally highk_(cat)/K_(M) value.

Cleavage of the S654A mutant fragments with the wild-typeenzymes—Proenzyme S654A mutant fragments were used as substrates ininvestigating autoactivation properties of the wild-type fragments. Theactive enzyme cleaves and activates proenzyme S654A mutant that has nocatalytic activity even in the two-chain form. Thus, the autoactivationcan be studied in a simple enzyme-substrate system. The activity ofwild-type SP, CCP2-SP, and CCP1-CCP2-SP on the S654A mutants of the SP,CCP2-SP, and CCP1-CCP2-SP fragments were measured in each combination.Cleavage of the S654A mutants was followed by reducing SDS-PAGE.Catalytic efficiency values are presented in Table 7. In allenzyme-substrate pairs a high catalytic efficiency, comparable to theactivities on synthetic ester substrates could be detected. SP fragmentactivated the three S654A mutants at a similar rate. The highestk_(cat)/K_(M) values were obtained for the self-activation of theCCP2-SP fragment (i.e. its activity on the S654A mutant CCP2-SP) (FIG.14).

4. Discussion of Results with the MASP-Fragments

A principal, but so far debated question is the autoactivation capacityof MASP-1 and MASP-2. Since prior to renaturation the MASP proteinfragments were single-chain polypeptides, the autoactivation process,which involves the cleavage of an Arg-Ile bond in the serine proteasedomain, could be followed on reducing SDS-PAGE. Upon renaturation theMASP-1 CCP1-CCP2-SP fragment became fully activated, and gave two bands(corresponding to the “γ”- and “B”-chains) on the PAG. In addition tothe two major bands, we observed a minor band of 24 kDa, whichco-purified with the MASP-1 CCP1-CCP2-SP fragment. This band is mostprobably an autolytic cleavage product of the serine protease domain.The autolytic cleavage at the Arg⁵⁰⁴-Asp⁵⁰⁵ bond removes the active siteHis, which results in the destruction of the protease activity. We canconclude that MASP-1 has a strong propensity to autoactivate and theactivated enzyme is prone to autodegradation upon prolonged incubation.The physiological relevance of this autolysis is yet unknown, but it isnoteworthy to mention that we observed the corresponding autolyticproduct of the full-length MASP-1 protein, in partially purified serumMBL-MASPs complexes (data not shown).

In terms of autoactivation capacity the MASP-2 catalytic fragmentsrevealed somewhat different features. Throughout the renaturationprocedure the MASP-2 fragments retained their proenzyme form. Postpurification, however, they migrated as two separate bands on reducingSDS-PAGE, a characteristic of activated enzyme. Subsequent N-terminalsequencing confirmed that the cleavage occurred at the Arg⁴⁴⁴-Ile⁴⁴⁵activation bond. Therefore, it is very likely that MASP-2 also has thecapacity to autoactivate, but the reaction is slow at lowconcentrations. Typically, to prevent aggregation the proteinconcentration is kept very low during renaturation (1-2 μg/ml). Duringpurification, however, the protein concentration increases dramaticallyon the ion exchange columns (the peak fractions could be as concentratedas 1-2 mg/ml), which can facilitate the autoactivation process andincrease the overall rate of active enzyme formation. We are tempted tobelieve that the autoactivation reaction of MASP-1 and MASP-2 is similarto that of C1r: In the first step zymogen molecules activate zymogens,while in the second step the generated active enzymes cleave zymogenmolecules. In a concentrated solution the probability of the reactiveencounters between MASP-2 molecules (either zymogenic or activated) isgreater than in a diluted solution. It was shown by Vamp-Jensen et al.that the MBL-MASP-2 complex alone is sufficient for complementactivation. Although, we do not know the stoichiometric composition ofthis complex, but it is very likely that at least two MASP-2 moleculesassociate with the MBL molecule (Chen, 2001). This view is furtherstrengthened by the fact that both MASP-1 and MASP-2 form homodimersthrough their N-terminal CUB1-EGF-CUB2 region (Thielens, 2001). Ourobservations reveal that the smaller MASP-2 fragments (CCP2-SP and SP)can also autoactivate, indicating that the autoactivating ability is aninherent property of the serine protease domain. In contrast to previoussuggestion (Rossi, 2001) it seems that the CCP modules of MASP-2 do notplay an essential role in this process. The MASP-2 CCP1-CCP2-SP fragmentalso showed a sign of autodegradation, since its N-terminal begins withIle²⁹¹ instead of the expected Ala. It looks very likely that the shortstretch of CUB2 domain fused with the Ala-Ser-Met tripeptide at theN-terminus (Ala-Ser-Met-Thr-Gly-Trp-Lys²⁹⁰) folded loosely and theLys²⁹⁰-Ile²⁹¹ bond in this region was an easy target for a protease withtrypsin-like specificity (i.e. MASP-2).

Probably the most controversial issue concerning the substratespecificities of MASP-1 and MASP-2 is their ability to cleave C3. Weaimed at resolving this debate by using our catalytic fragments andcarefully prepared C3 substrates. C3 contains a thiol ester group insidethe molecule that becomes exposed after the cleavage of C3 by the C3convertase enzymes. The exposed thiol ester group is then rapidlyhydrolyzed or reacted with a nucleophile on the cell surface.Nevertheless, uncleaved C3 is also prone to spontaneous hydrolysisyielding non-functional C3(H₂0), which may occur during the purificationprocess and upon prolonged storage. It is important to differentiatebetween ‘live’ C3 (i.e. C3 with an intact thiol ester bond) and ‘dead’C3 (C3 with a reacted thiol ester bond), as they respond differently toproteolysis although they migrate similarly on SDS-PAGE. We measured thekinetic parameters of C3 cleavage by using both freshly prepared ‘live’C3 and ‘dead’ C3. Our results demonstrate that the k_(cat)/K_(M) valuesof both MASP-1 and MASP-2 on ‘live’ C3 substrate were very low (˜300M⁻¹s⁻¹). We believe that this marginal activity is not sufficient fordirect complement cascade activation in the presence of such potentinhibitors in serum as C1-inhibitor or alpha-2-macroglobulin. ‘Dead’ C3was cleaved with a low but significant efficiency: the k_(cat)/K_(M)values were about 10-20 fold higher than in the case of ‘live’ C3cleavage. It is possible therefore that the C3 cleaving activities ofthe MASPs, reported earlier in the literature, were mostly due to thehigh ratio of ‘dead’ C3 in C3 preparations. Still, it can not beexcluded that the marginal activities of MASP-1 and MASP-2 on ‘live’ C3may have physiological consequences (e.g. initiating the alternativepathway). Nevertheless, we strongly believe that C3 is not the realnatural substrate of either MASP-1 or MASP-2.

C4, similarly to ‘live’ C3, was basically resistant against theproteolytic activity of MASP-1. C2, however, was digested by therecombinant MASP-1 fragment at a moderate rate. The k_(cat)/K_(M) valuefor the C2 cleavage was two orders of magnitude higher, than that of theC4 cleavage. The C2 cleavage alone, however, is not sufficient toinitiate the complement cascade, since physiologically relevant C2cleavage occurs on the C2C4b complex. It should also be stressed thatthis moderate k_(cat)/K_(M) value is smaller by an order of magnitudethan the corresponding values of MASP-2 or C1s.

The recombinant MASP-2 fragments cleaved C2 and C4 efficiently. This andthe autoactivating capacity of MASP-2 is in accordance with theobservation that MBL-MASP-2 complex can activate the complement cascade.Since we expressed three different functionally active truncatedcatalytic fragments of MASP-2 (i.e. CCP1-CCP2-SP, CCP2-SP and SP), wecould analyze the role of the individual domains in C2 and C4 cleavage.It can be concluded that C2 cleavage is mediated entirely by the serineprotease domain. The highest k_(cat)/K_(M) value was measured in thecase of the single SP domain fragment. The addition of the CCP modulesto the SP domain somewhat decreased the catalytic efficiency. The SPdomain therefore probably contains all necessary contact sites forefficient C2 binding and cleavage, and the CCP domains do not contributeto this reaction. On the contrary, C4 digestion of the MASP-2 fragmentsis influenced by the presence of the CCP domains. Although, the singleSP domain can cleave C4 at a moderate efficiency, the addition of theCCP2 module to the SP domain increases the k_(cat)/K_(M) valuedramatically (44-fold increase). It seems very likely, that the CCP2domain contains additional binding site(s) for the protein substrate C4.This is reflected in the decrease of the K_(M) value (from 2.0 μM to 0.4μM), which indicates a stronger binding of the substrate. Our resultsare similar to previous results of Rossi et. al., who demonstrated theessential role of CCP2 module in the C4 digestion ability of C1s (Rossi,1998). Apparently, the CCP2 domains play an essential role indetermining the enzymatic properties of the MASP-2 proteases (Table 4).The presence of the CCP1 module on the CCP1-CCP2-SP fragment of MASP-2decreases its catalytic efficiency. Nevertheless, the catalyticefficiency of MASP-2 CCP1-CCP-2-SP against C4 is still high, and thek_(cat)/K_(M) values of this MASP-2 fragment are practically equal (˜500000 M⁻¹s⁻¹) for the C2 and C4 substrates.

The above results, together with the finding that MASPs are inactiveagainst C3 provide an assay method for selective determination of MASP-1and MASP-2 levels. Such an assay would comprise for example the steps of

-   -   monitoring C2 cleavage and C4 cleavage by MASP proteins in        aliquots of the sample whereas, if desired, other complement        pathways are blocked,    -   considering C4 conversion as a result of MASP 2 activity and C2        conversion as a result of MASP-1 and MASP-2 activity together    -   calculating MASP-1 and MASP-2 levels using either known specific        activity values of said proteins or CCP1-CCP2-SP fragments as        inner standards.

The question was raised whether a similar assay method can be carriedout on the basis of different inhibitor specificities of MASP-1 andMASP-2.

Both C1-inhibitor and alpha-₂M were able to block the proteolyticactivities of both MASP-1 and MASP-2. In the case of C1-inhibitor a 1:1molar ratio was necessary for complete inhibition. The observed pseudofirst order rates of reaction (k_(obs)) with C1-inhibitor were 5-foldless for the MASP-1 CCP1-CCP2-SP than for the MASP-2 CCP1-CCP2-SPfragment. Keeping in mind that we have measured a 4-5 fold higherspecific activity for MASP-1 than for MASP-2 for arginine at the P1sites by the competing oligopeptide substrate library assay, this resultindicates that MASP-2 has much better interaction properties withC1-inh. The K_(i) values for the C1-inhibitor and MASP-2 CCP1-CCP2-SPreaction were in the nanomolar range, which is an order of magnitudeless than those of the C1r and C1s interactions with C1-inhibitor and isan indicator of strong binding (Sim, 1980) (Table 4).

Thus, C1-inhibitor specificity of MASP-2 is not sufficient todifferentiate between the two MASPs.

However, a marginal molar excess of alpha-2-macroglobulin was enough tocompletely block the proteolytic activity of MASP-1, whereas only a40-fold molar excess over the MASP-2 fragments was capable ofsignificantly hindering C2 and C4 cleavage. The results of theexperiment, where alpha-2-macroglobulin and C1-inhibitor competed foreither the MASP-1 or the MASP-2 catalytic fragment, showed clearly thatC1-inhibitor is a better inhibitor of MASP-2, while alpha-₂M is asignificant inhibitor of MASP-1. In this respect it is interesting tonote that MASP-1 represents an evolutionary ancient type of the serineproteases, since the active site Ser⁶⁴⁶ is encoded by a TCN type codon(Brenner, 1988). Another highly conserved Ser residue downstream theactive site (Ser⁶⁶⁷) is also encoded by TCN (Krem, 2001), moreover,there is a histidine loop around the active site and the SP domain isencoded by six exons (Endo, 1998). Alpha-2-macroglobulin, which isrelated to C3, also belongs to the thiol ester group of proteins.

Thus, an assay method for assessing MASP-1 and MASP-2 levels in a sampleof biological origin can be carried out by performing the followingsteps:

-   -   monitoring C2 cleavage in the sample and considering C2        conversion as a result of MASP-1 and MASP-2 activity together,    -   adding a calculated amount of α2M to the sample to inhibit        MASP-1 activity (e.g. MASP-1 activity can be “titrated” to zero        by following the curve for C2 conversion) but to leave MASP-2        activity unchanged or changing it to a negligible or a        calculable extent,    -   monitoring C2 activity in the sample comprising α2M,    -   calculating MASP-1 and MASP-2 levels using either known specific        activity values of said proteins or CCP1-CCP2-SP fragments as        inner standards.

By comparing the substrate specificities of MASP-1 and MASP-2 on smallsynthetic peptide substrates we have created a screening method forsubstrates of these MASP enzymes. To the best of our knowledge thepresent invention provides the first comprehensive analysis of thesubstrate specificities and relative activities of MASP-1 and MASP-2using a competing oligopeptide substrate library. The most importantfinding is that MASP-1, besides being a very potent peptidase, shows anextreme Arg selectivity at the P1 site of the substrates. While undersame conditions the consumption of the P1 Lys substrate is quitecomparable between the MASP-1 and MASP-2 catalytic fragments, the MASP-1CCP1-CCP2-SP fragment digests the P1 Arg peptide 4-5 fold moreefficiently than the corresponding fragment of MASP-2. The substratespecificity of MASP-2 resembles trypsin, whereas MASP-1 acts likethrombin or mouse endoproteinase Arg-C (Antal, 2001). The high catalyticpotential of MASP-1 suggests that there exists a natural substrate forMASP-1, which is cleaved more efficiently than C3. Indeed, in recentstudies Hajela at al. showed that MASP-1 cleaves fibrinogen and FactorXIII at much faster rates than C3 and is thus, similarly to thrombin,able to catalyze the formation of cross-linked fibrin.

Using the results of the present study and data obtained by otherlaboratories we can compare some basic characteristics of the MASP-1 andMASP-2 proteases (Table 4). All of these proteases are capable ofautoactivating. These proteins (MASP-1 and MASP-2) circulate in serum ashomodimers, which facilitates their autoactivation, as autoactivationpresumes contact between two serine protease domains. In fluid phase,however, MASP-2 autoactivates only at higher concentrations, thanMASP-1. The capacity to autoactivate enables MASP-2 through cleaving C2and C4 to initiate the complement cascade after having received anactivation signal from a recognition molecule (e.g. C1q, MBL orficolin). As in vivo natural substrates of MASP-1 are not yet wellcharacterized the physiological consequences of its autoactivatingcapacity is hard to predict.

The isolated serine protease domains of C1r, C1s and MASP-2 possessproteolytic activities. In the case of C1s and MASP-2 the serineprotease domains alone cleave C2 as efficiently as the whole molecule.Nevertheless, for efficient C1s cleavage by C1r and efficient C4cleavage by C1s and MASP-2 the CCP2 module is essential. This could bepartly attributed to the feature conserved during evolution that theCCP2 module forms a compact structural and functional unit with the SPdomain (Gaboriaud, 1998/Gaboriaud, 2000/Budayova-Spano, 2002). All fourproteases can be inhibited by the serpin C1-inhibitor, but MASP-1 ismuch more sensitive to alpha-₂M.

Our work provided an important detail in the identification of thenatural substrate of MASP-1. We showed that MASP-1 is a very potentpeptidase and has an extreme Arg specificity at the P1 site. Thisbehavior resembles thrombin, the terminal protease of the blood clottingcascade,

Several lines of evidences indicate that the complement and the bloodclotting cascades evolved from a common ancestral protease cascade(Krem, 2002). It has also been reported that sequence homologues offibrinogen served immunological roles. Therefore, it can be speculatedthat MASP-1, as an ancient protease, shows characteristics of bothcascades (complement-like features: interaction with MBL, inhibition byC1-inhibitor; clotting-like feature: arginine selectivity at P1,cleavage of fibrinogen and Factor XIII).

Thus, it is contemplated that MASP-1 can be used for inducing formationof fibrin from fibrinogen and thereby promoting blood clotting.

5. Discussion of Results with the C1r Fragments

In the present study we used an E. coli based expression system forrecombinant production of fragments representing the catalytic region ofhuman C1r.

Previously the baculovirus-insect cell system was used to producerecombinant C1r and C1s and their fragments (14, 20, 21). In this systemthe post-translational modifications are not complete. Furthermore, thelevel of 13-hydroxylation of the Asn residue in the EGF domain (Asn¹⁶⁷in C1r and Asn¹⁴⁹ in C1s) is very low (14, 22) and the glycosylationpatterns differ significantly from the complex type glycosylation foundin the case of serum proteins. These differences in the posttranslational modifications however did not seem to affect significantlythe functional properties of the recombinant proteins; at least somestudies showed that aglycosylated proteins retained their biologicalactivity (14, 21).

In order to explore the contribution of the individual domains to theabove-mentioned properties of C1r we successively deleted the CCPdomains preceding the SP domain from the cDNA. As a result, we made andexpressed three cDNA constructs. The recombinant proteins accumulated asinclusion bodies inside the E. coli cells. After disruption of the cellswe purified and renatured these proteins. The renatured fragments werepurified to homogeneity by ion-exchange chromatography and gelfiltration. After these procedures all the three fragments were in acorrectly folded, functionally active form as confirmed by subsequentphysico-chemical and enzymatic measurements. After renaturation thewild-type fragments were present in the activated, two-chain form.Autoactivation is a good indicator of correct folding. Indeed, by theend of the renaturation procedure the activation was complete. To studythe zymogen form of the enzymes, as well as the autoactivationprocedure, we constructed and expressed mutant C1r fragments i.e. stableproenzymes. In one set of experiments we mutated the Arg⁴⁶³-Ile⁴⁶⁴cleavage site to Gln-Ile (27), while in another set we changed thecatalytic site Ser⁶⁵⁴ to Ala. In the first case the zymogen cannot beactivated by a trypsin-like serine protease, however it could be cleavedand converted to an active enzyme by thermolysin. In the case of theSer654Ala mutants the Arg-Ile bond can be cleaved by wild-type C1r, butthe mutant itself cannot function as an active protease. All of theseC1r fragments were produced with a similar yield except the CCP2-SPfragments where the renaturation efficiency was significantly higher.This can be interpreted as the CCP2 domain closely associates with theSP domain forming a compact cooperative folding unit. The SP domainalone is less stable as it is indicated by the DSC measurements. TheCCP1-CCP2-SP fragment contains the CCP1 module, which associates looselyto the CCP2-SP. The larger, more complex structure of CCP1-CCP2-SP canaccount for its lower renaturation yield.

The DSC measurements indicate that all of the renatured fragments havecompact, folded structures. The calorimetric heat denaturation curves(excess heat capacity against temperature) show cooperative unfolding ofthe native structures in the case of the three fragments. The heatcapacity curve of the recombinant CCP1-CCP2-SP is essentially identicalto the curve of CCP1-CCP2-SP expressed in the baculovirus insect cellsystem. According to the DSC curves the SP fragment has the least stablestructure. The CCP2 module exerts a dramatic stabilizing effect, as themidpoint of the heat denaturation peak of SP (47.5° C.) is being shiftedto 55.4° C. in the case of the CCP2-SP fragment (FIG. 12). The presenceof the CCP1 module stabilizes the structure further, but this effect ismuch less significant than that of CCP2. These results are in agreementwith the homology model of the catalytic region of C1r (8) and with thecrystal structure of the CCP2-SP fragment of C1s (28). According to thecrystal structure the CCP2 module associates to SP domain through arigid module-domain interface involving intertwined proline andtyrosine-rich polypeptide segments. Such a rigid CCP-SP assembly isconserved in other extracellular proteases (29). Gel filtrationexperiments show that the SP and CCP2-SP fragments are monomers at allpH values, whereas the CCP1-CCP2-SP fragment is a dimer at neutral andbasic pH (FIG. 11). Like serum C1r (30), our recombinant CCP1-CCP2-SPdimers dissociate under slightly acidic conditions. These resultsprovide straightforward experimental evidence that the CCP1 domain isinvolved in the dimerization of C1r. Previously a three-dimensionalmodel of activated (γB)₂ has been constructed, which was based onchemical cross-linking and homology modeling (8). Chemical cross-linkingof the (γB)₂ fragment produced by autolytic cleavage of the active serumC1r indicated the existence of converse salt bridges between Lys²⁹⁹ inthe N-terminal region of the y segment of one monomer, and Glu⁵¹⁰ of theserine protease domain of the other monomer. Our CCP1-CCP2-SP constructhowever begins with the Cys³⁰⁹ of C1r preceded by the Ser-Thr-Gln-AlaN-terminal fusion peptide and therefore lacks Lys²⁹⁹. The fact that ourrecombinant CCP1-CCP2-SP is a stable dimer indicates that formation ofsalt bridges between Lys²⁹⁹ and Glu⁵¹⁰ is not a prerequisite ofdimerization. Future site directed mutagenesis experiments could revealthe amino acid residues of the CCP1 module that are involved in C1rdimerization.

Regarding enzymatic activity it is surprising that the serine proteasedomain of C1r alone can cleave C1s with a rate comparable with that ofthe activation by the CCP1-CCP2-SP fragment. This indicates that the SPdomain contains all the structural elements necessary for C1s bindingand cleavage. The presence of the CCP2 domain alone in the CCP2-SPfragment however causes a dramatic increase (one order of magnitude) ofthe k_(cat)/K_(m) value of the reaction. We can conclude that the CCP2domain is responsible for the enhancement of the efficiency of the C1scleaving activity of C1r. We suggest that although the SP domain alonecan cleave C1s, the CCP2 domain provides with additional contactsurfaces for binding and orienting the substrate. As we mentioned above,our results obtained by DSC indicate that the CCP2 domain stronglystabilizes the structure of the SP domain. The increase of thek_(cat)/K_(m) value could be explained in principle by thisdomain-domain interaction. The similar esterolytic activity of therecombinant C1r fragments however shows that this is not the case. It isobvious that all the three recombinant C1r fragments contain a fullyfunctional active site. The changes in stability caused by the additionof CCP modules to the serine protease domain do not affect the catalyticpower of the serine protease active site on the synthetic substrates. Wesuspect that the dramatic increase of the C1s cleaving ability of theCCP2-SP fragment is due to additional substrate binding sites present onthe surface of the CCP2 domain, and not to the stabilizing effect. Thecorresponding k_(cat)/K_(m) value of the CCP1-CCP2-SP fragment for C1scleavage however is smaller than that of the CCP2-SP fragment. As weshowed above the CCP1 domain is responsible for the dimerization of C1r.The catalytic site of C1r or the substrate binding residues on the CCP2domain can be less accessible for the C1s in the CCP1-CCP2-SP dimersthan in the CCP2-SP and SP monomers. It is also very likely that theCCP1 domain does not contain additional substrate binding sites for C1s.We should keep in mind that we are dealing with fluid phase reactions inour present study. Inside the C1 complex however the catalytic domainsof C1r and C1s are precisely positioned, therefore the efficiency of C1scleavage by the C1r dimer can be significantly higher (31).

Our work with recombinant fragments provided valuable informationconcerning the autoactivation of C1r. The S654A mutant retains itszymogen form during the renaturation and purification procedure, whereasthe wild-type C1r fragments are fully activated after the sametreatment. We can conclude that this activation is a true autoactivationand that extrinsic (i.e. E. coli) proteases do not contribute to it.Since all the three wild-type fragments autoactivate, two importantconclusions can be drawn: (i) dimerization is not a prerequisite forautoactivation, (ii) autoactivation is an inherent property of theserine protease domain. Previously, autoactivation was shown to be aproperty of the dimers both in the case of the entire molecule and itsCCP1-CCP2-SP fragment (7). Under acidic conditions (pH<5.5) the C1rdimer dissociates and the resulting monomers lose their ability ofautoactivation (32). Since at acidic pH the catalytic activity of theserine protease active center is expected to decrease it was not clearwhich phenomenon was responsible for the loss of the autoactivationability. Our results indicate that under physiological conditions themonomeric CCP2-SP fragment is capable of autoactivation i.e.dimerization is not required for autoactivation. Since the SP domainitself retains the ability to autoactivate, the presence of even one CCPdomain is not a prerequisite for autoactivation.

Autoactivation of C1r is supposed to be a two step process. In theinitial step zymogen molecules activate zymogens, while in the secondstep the generated active enzymes cleave zymogen molecules. The factthat our wild-type fragments can autoactivate shows the existence of theinitial step. The second step could be studied in detail using ourzymogen mutants. The Ser654Ala mutants, which have an inactive catalyticcenter but have a cleavable Arg-Ile bond were used as substrates for thewild-type fragments. We determined the kinetic constants for these typeof reactions (Table 7). Each Ser654Ala mutant was cleaved by its ownwild-type counterpart. The SP fragment showed effective self-cleavageability. The higher catalytic efficiency of the CCP2-SP constructcompared to that of the SP domain can be interpreted assuming that theCCP2 domain, like in the case of the C1s cleavage, orients the Arg-Ilebond of one C1r in a favorable position to be cleaved by the active siteof the other C1r. The CCP 1-CCP2-SP fragment possess significantly lowerk_(cat)/K_(m) values than the other two fragments. Since on(CCP1-CCP2-SP)₂ we follow intermolecular (inter dimeric) cleavage, wemay conclude that dimer formation partially blocks the accessibility ofeither the catalytic site of the protease, or the activation site of theproenzyme and therefore decreases the efficiency of the proteolysis. Toclarify this question we carried out experiments with combinations ofthe different fragments. The fact that the wild-type SP fragment exertssimilar proteolytic activity on the dimeric CCP1-CCP2-SP fragment and onthe smaller monomer fragments, indicates that the Arg-Ile bond to becleaved is accessible for extrinsic cleavage. The catalytic efficiencyof CCP2-SP on CCP1-CCP2-SP also supports this observation. In acomplementary experiment the wild-type CCP1-CCP2-SP fragment showeddecreasing catalytic efficiency with the increasing size of thesubstrate proenzyme SP, CCP2-SP and CCP1-CCP2-SP S654A fragments. It isvery likely that the catalytic site of one CCP1-CCP2-SP is pointed tothe “inside” (i.e. toward the other CCP1-CCP2-SP molecule) in the dimerand this positioning facilitates the intramolecular autolytic cleavage.

The major conclusion of this work is that the serine protease moduleitself is an autonomous folding unit with inherent serine proteaseactivity similar to that of intact C1r. The SP module has the ability tocleave C1s, the natural substrate of C1r, and autoactivation property isalso retained.

Comparative measurements highlighted the role of the CCP modules in C1ras modulators of the catalytic functions through allosteric effectsoccurring upon binding to natural substrates and dimerization. Theintimate interaction of the SP domain with the CCP2 domain is reflectedin the sizeable stabilizing effect observed if CCP2 is attached to theSP module.

Antibody Production

Once folded C-terminal fragments have been obtained it becomes possibleto prepare antibodies, preferably monoclonal antibodies against thesefragments. Such techniques are well known in the art and described e.g.in Bonifacino et al. (2000), which is incorporated herein by reference.Monoclonal antibodies are useful then for detection of the multidomainserine proteases themselves. Moreover, they can be used in assay kits orin diagnostic kits for detection of e.g. MASP, C1r or C1s levels inbiological samples. If the antibodies are directed to substrate bindingsites they may serve as inhibitors as well.

Tables

Kinetic Constants for the Cleavage of Live C3, dead C3 (Table 1), C2 andC4 (Table 2) by MASP-1γB and MASP-2 Fragments

TABLE 1 Cleavage of C3 live C3 C3(NH₃) k_(cat)/K_(M) (M⁻¹ s⁻¹)k_(cat)/K_(M) (M⁻¹ s⁻¹) MASP-1γB 300 ± 30 6 100 ± 600 MASP-2γB 350 ± 203 300 ± 300 ^(a)values indicated are the averages of 2 to 4 experiments,standard errors are indicated below actual values.

Both MASP-1γB and MASP-2γB exhibited marginal proteolytic activitytowards intact C3 (live C3). When inactivated C3 (C3(NH₃)) wassubstituted for intact C3 the reactivity of the MASPγBs increased about10-20 fold.

TABLE 2 C2 and C4 cleavage C2 C4 k_(cat) K_(M) k_(cat)/K_(M) k_(cat)K_(M) k_(cat)/K_(M) (s⁻¹) (μM) (M⁻¹ s⁻¹) (s⁻¹) (μM) (M⁻¹ s⁻¹) MASP-1γB0.10 ± 0.07 4.8 ± 4.3 30 000 ± 12 000 0.002 ± 0.001 2.7 ± 2.4 690 ± 400MASP-2γB 1.9 ± 0.8 4.0 ± 1.7 500 000 ± 9 000  0.9 ± 0.4 1.6 ± 0.5 550000 ± 50 000  MASP-2 CCP-SP 3.0 ± 2.2 2.7 ± 2.0 1 300 000 ± 200 000  2.0 ± 1.0 0.4 ± 0.2 5 700 000 ± 550 000   MASP-2 SP 3.9 ± 3.0 3.2 ± 2.82 500 000 ± 1 300 000 0.20 ± 0.06 2.0 ± 1.0 130 000 ± 30 000  ^(a)valuesindicated are the averages of 2 to 4 experiments, standard errors areindicated below actual values

The MASP-2 fragments showed substantial proteolytic activity in both C2and C4 cleavage. MASP-1γB cleaved C2 at a low but significant rate,whereas C4 cleavage was negligible.

TABLE 3 Kinetic constant for the reaction of MASP-1γB or MASP-2γB withC1-inh k_(obs) (s⁻¹)^(a) K_(i) (nM) k₂ (s⁻¹) MASP-1γB 1.4 × 10⁻³^(b)N.A. ^(b)N.A. MASP-2γB 7.8 × 10⁻³ 8.8 8 × 10⁻³ ^(a)the pseudo-firstorder rate of reaction at [C1-inh] = 350 nM ^(b)data not available

TABLE 4 Comparison of the lectin and the classical pathway serineproteases with respect to various properties Feature Enzymesautoactivation capacity MASP-1 > MASP-2 C2 cleaving capacity MASP-2 >>MASP-1 C4 cleaving capacity MASP-2 C3 cleaving capacity(MASP-1~MASP-2)^(a) C1-inhibitor sensitivity MASP-2 > MASP-1 α2Msensitivity MASP-1 > MASP-2 ^(a)only to a very limited extent and on“dead” C3 substrate

TABLE 5 Catalytic efficiency of wild-type C1r fragments and thethermolysin activated R463Q mutants for synthetic substrates^(a) SPCCP2-SP CCP1-CCP2-SP Z-Lys-S-Bzl  1600^(b)  1900^(b)  1300^(b)Z-Gly-Arg-S-Bzl 164000^(b) 210000^(b) 174000^(b) Z-Gly-Arg-S-Bzl112000^(c) 147000^(c) 124000^(c) ^(a)k_(cat)/K_(M) (s⁻¹M⁻¹). Measurementwere carried out in 20 mM Tris, 145 mM NaCl, pH 7.4, at 30° C.k_(cat)/K_(M) values are the averages of at least 3 independentexperiments. ^(b)Wild-type C1r fragments ^(c)C1r R463Q fragments, aftera treatment with 10 U thermolysin (Sigma)/mg C1r fragment, pH 8.0, 30°C., 2 h.

TABLE 6 Efficiency of C1s cleavage^(a) SP 28000 ± 2000 CCP2-SP 208000 ±10000 CCP1-CCP2-SP 58000 ± 4000 ^(a)k_(cat)/K_(M) (s⁻¹M⁻¹). Reactionswere performed in 20 mM Tris, 145 mM NaCl, pH 7.4, at 30° C.

TABLE 7 Self-cleavage efficiency of the C1r fragments^(a) CCP1-CCP2-SPSP S654A CCP2-SP S654A S654A SP 71000 75000 72000 CCP2-SP 68000 13000074000 CCP1-CCP2-SP 45000 15900 6200 ^(a)k_(cat)/K_(M) (s⁻¹M⁻¹). TheS654A inactive zymogen mutants were used as substrates for the wild-typefragments. Reactions were performed in 20 mM Tris, 145 mM NaCl, pH 8.3,at 37° C. The values are the averages of 2-4 independent measurements.Variation between the results of the individual measurements was lessthan 10%.

The multidomain complement serine protease fragments of the inventionare useful for a variety of purposes, e.g. as research tools incomplement research,

-   -   as standards or controls in several type of assay methods for        assessing multidomain complement serine protease,    -   for raising antibodies against multidomain complement serine        proteases,    -   for screening substrates or inhibitors of multidomain complement        serine protease,    -   for treatment, diagnosis and/or research of complement related        diseases.

REFERENCES

-   Adachi et al., Trans. Proc. 19(1): 1145 (1987)-   Ahrenstedt et al., New Engl. J. Med. 322:1345-9 (1990)-   Antal, J., G. Pál, B. Asbóth, Zs. Buzás, A. Patthy, and L.    Gráf. 2001. Specificity assay of serine proteinases by reverse-phase    high-performance liquid chromatography analysis of competing    oligopeptide substrate library. Anal. Biochem. 288:156.-   Arlaud, G. J., and N. M. Thielens. 1993. Human complement serine    proteases C1r and C1s and their proenzymes. Methods Enzymol. 223:61.-   Arlaud, G. J., C. L. Villiers, S. Chesne, and M. G. Colomb. 1980.    Purified proenzyme CI r. Some characteristics of its activation and    subsequent proteolytic cleavage. Biochim. Biophys. Acta 616:116.-   Arlaud, G. J., J. Gagnon, C. L. Villiers, and M. G. Colomb. 1986.    Molecular characterization of the catalytic domains of human    complement serine protease C1r. Biochemistry 25:5177.-   Arlaud, G. J., M. G. Colomb, and J. Gagnon. 1987. A functional model    of the human C1 complex. Immunol. Today 8:106.-   Arlaud, G. J., S. Chesne, C. L. Villiers, and M. G. Colomb. 1980. A    study on the structure and interactions of the C1 sub-components C1r    and C1 s in the fluid phase. Biochim. Biophys. Acta 616:105.-   Aulak, K. S., V. H. Donaldson, M. Coutinho and A. E. Davis, 3^(rd)    (1993). C1-inhibitor: structure/function and biologic role. Behring    Inst Mitt: 204-13.-   J. S. Bonifacino, M. Dasso, J. B. Harford, J.    Lippincott-Schwartz, K. M. Yamada: Current Protocols In Cell Biology    Volume 2, Chapter 16 (2000) John Wiley & Sons, Inc. NY, US.-   Brenner, S. 1988. The molecular evolution of genes and proteins: a    tale of two serines. Nature 334:528.-   Budayova-Spano, M., M. Lacroix, N. M. Thielens, G. J. Arlaud, J. C.    Fontecilla-Camps, and C. Gaboriaud. 2002. The crystal structure of    the zymogen catalytic domain of complement protease C1r reveals that    a disruptive mechanical stress is required to trigger activation of    the C1 complex. EMBO J. 21:231.-   Castillo, M. J., K. Nakajima, M. Zimmerman, and J. C. Powers. 1979.    Sensitive substrates for human leukocyte and porcine pancreatic    elastase: a study of the merits of various chromophoric and    fluorogenic leaving groups in assays for serine proteases. Anal.    Biochem. 99:53.-   Chen, C.-B., and R. Wallis. 2001. Stoichiometry of complexes between    mannose-binding protein and its associated serine proteases:    Defining functional units for complement activation. J. Biol. Chem.    276:25894.-   Chenoweth et al., Complement. Inflamm. 3:152-165 (1981)-   Chenoweth et al., Complement 3:152-165 (1986)-   Clark, E. B. (1998) Refolding of recombinant proteins. Current    Opinion in Biotechnology 9, 157-163.-   Collaborative Computational Project, Number 4. Ada Cryst. D50, 760    (1994)-   Collard C D, Vakeva A, Morrissey M A, Agah A, Rollins S A, Reenstra    W R, Buras J A, Meri S, Stahl G L. 2000. Complement activation after    oxidative stress: role of the lectin complement pathway. Am J Pathol    156(5):1549-56.-   Dahl, M. R., S. Thiel, M. Matsushita, T. Fujita, A. C. Willis, T.    Christensen, T. Vorup-Jensen, and J. C. Jensenius. 2001. MASP-3 and    its association with distinct complexes of the mannanbinding lectin    complement activation pathway. Immunity 15:127.-   Demling et al., Surgery 106:52-9 (1989)-   Deppisch et al., Kidney Inst. 37:696-706 (1990)-   Dobó, J., P. Gál., K. Szilágyi, S. Cseh, Z. Lörincz, V. N.    Schumaker, and P. Závodszky. 1999. One active C1r subunit is    sufficient for the activity of the complement C1 complex:    stabilization of C1r in the zymogen form by point mutations. J.    Immunol. 162:1108.-   Dodds, A. W. (1993). Small-scale preparation of complement    components C3 and C4. Methods Enzymol 223: 46-61.-   Dumestre-Perard C, Ponard D, Drouet C, Leroy V, Zarski J P, Dutertre    N, Colomb M G. 2002. Complement C4 monitoring in the follow-up of    chronic hepatitis C treatment. Clin Exp Immunol. 127(1):131-6.-   Endo, Y., M. Takahashi, M. Nakao, H. Saiga, H. Sekine, M.    Matsushita, M. Nonaka, and T. Fujita. 1998. Two lineages of    mannose-binding lectin-associated serine protease (MASP) in    vertebrates. J. Immunol. 161:4924.-   Fraker, P. J. and J. C. Speck, Jr. (1978). Protein and cell membrane    iodinations with a sparingly soluble chloroamide,    1,3,4,6-tetrachloro-3a,6a-diphrenylglycoluril. Biochem Biophys Res    Commun 80: 849-57.-   French, G. S., Wilson, K. S. Acta Cryst. A34, 517 (1978)-   Gaboriaud, C., V. Rossi, I. Bally, G. J. Arlaud, and J. C.    Fontecilla-Camps. 2000. Crystal structure of the catalytic domain of    human complement C1s: a serine protease with a handle. EMBO J.    19:1755.-   Gaboriaud, C., V. Rossi, J. C. Fontecilla-Camps, and G. J.    Arlaud. 1998. Evolutionary conserved rigid module-domain    interactions can be detected at the sequence level: the examples of    complement and blood coagulation proteases. J. Mol. Biol. 282:459.-   Gál, P., and G. Ambrus. 2001. Structure and function of complement    activating enzyme complexes: C1 and MBL-MASPs. Curr. Prot. Pept.    Sci. 2:43.-   Gál. P_, M. Sárvári, K. Szilágyi, P. Závodszky, and V. N.    Schumaker. 1989. Expression of hemolytically active human complement    component C1r proenzyme in insect cells using a baculovirus vector.    Complement Inflamm. 6:433.-   Gelfand et al, J. Clin. Invest. 70:1170 (1982)-   Gill, S. C., and P, H. von Hippel. 1989. Calculation of protein    extinction coefficients from amino acid sequence data. Anal.    Biochern. 182:319.-   Gráf, L., C. S. Craik, A. Patthy, S. Roczniak, R. J. Fletterick,    and W. J. Rutter. 1987. Selective alteration of substrate    specificity by replacement of aspartic acid-189 with lysine in the    binding pocket of trypsin. Biochem. 26:2616.-   Gulati, S., K. Sastry, J. C. Jensenius, P. A. Rice, and S.    Ram. 2002. Regulation of the mannan-binding lectin pathway of    complement on Neisseria gonorrhoeae by C1-inhibitor and    α₂-macroglobulin. J. Immunol. 168:4078.-   Guttman, Transplantation 17:383 (1974)-   Hack et al., Am. J. Med. 86:20-26 (1989)-   Hajela, K., J. Ferluga, G. Ambrus, P. Gal, K. Whaley, W. J.    Schwaeble, and R. B. Sim. 2002. The mannose-binding lectin-MASPs    system: a link between complement and the defensive role of    coagulation. Submitted-   Ji, Y-H., T. Fujita, H. Hatsuse, A. Takahashi, M. Matsushita, and M.    Kawakami. 1993. Activation of the C4 and C2 components of complement    by a proteinase in serum bactericidal factor, Ra reactive factor. J.    Immunol. 150:571.-   Jordan J E, Montalto M C, Stahl G L. 2001. Inhibition of    mannose-binding lectin reduces postischemic myocardial reperfusion    injury. Circulation 104(12):1413-8-   Kardos, J., P. Gál, L. Szilágyi, N. M. Thielens, K. Szilágyi, Zs.    Lörincz, P. Kulcsár, L. Gráf, G. J. Arlaud, and P. Závodszky. 2001.    The role of the individual domains in the structure and function of    the catalytic region of a modular serine protease, C1r. J. Immunol.    167:5202.-   Kawasaki, N., T. Kawasaki, and I. Yamashina. 1989. A serum lectin    (mannan-binding protein) has complement-dependent bactericidal    activity. J. Biochem. 106:483.-   Kissinger, C. R., Gehlhaar, D. K., Fogel, D. B. Acta Cryst. D55, 484    (1999)-   Kleiner D. E. and Stetler-Stevenson W. G. 1994. Quantitative    zymography: detection of picagram quantities gelatinases. Anal    Biochem 218(2):325-   Knechtle et al., J. Heart Transplant 4(5):541 (1985)-   Kojima et al., Nippon Jenzo Gakkai Shi 31:91-7 (1989)-   Krem, M. M., and E. Di Cera. 2001. Molecular markers of serine    protease evolution. EMBO J. 20:3036.-   Krem, M. M., and E. Di Cera. 2002. Evolution of enzyme cascades from    embryonic development to blood coagulation. TIBS. 27:67.-   Lacroix, M. B., C. A. Aude, G. J. Arlaud, and M. G. Colomb. 1989.    Isolation and functional characterization of the proenzyme form of    the catalytic domains of human C1r. Biochem. J. 257:885.-   Lacroix, M., C. Ebel, J. Kardos, J. Dobó, P. Gál, P.    Závodszky, G. J. Arlaud, and N. M. Thielens. 2001. Assembly and    enzymatic properties of the catalytic domain of human complement    protease C1r. J. Biol. Chem. 276:36233.-   Lacroix, M., V. Rossi, C. Gaboriaud, S. Chevallier, M.    Jaquinod, N. M. Thielens, J. Gagnon, and G. J. Arlaud. 1997.    Structure and assembly of the catalytic region of human complement    protease C1r: A three-dimensional model based on chemical    cross-linking and homology modeling. Biochemistry 36:6270.-   Laemmli, U. K. 1970. Cleavage of structural proteins during the    assembly of the head of bacteriophage T1. Nature 227:680.-   Laich, A. and R. B. Sim (2001). Complement C4bC2 complex formation:    an investigation by surface plasmon resonance. Biochim Biophys Acta    1544: 96-112.-   Leslie, A. G. W. in Crystallographic Computing, Oxford University    Press, Oxford (1990)-   Leytus, S. P., K. Kurachi, K. S. Sakariassen, and E. W. Davie. 1986.    Nucleotide sequence of the cDNA coding for human complement C1r.    Biochemistry 25:4855.-   Liota, L. A. and Stetler-Stevenson W. G. 1990 Cancer Biology, 1:96-   Lörincz, Zs., P. Gál, J. Dobó, S. Cseh, K. Szilágyi, G. Ambrus,    and P. Závodszky. 2000. The cleavage of two C1s subunits by a single    active C1r reveals substantial flexibility of the C1s-C1r-C1r-C1s    tetramer in the C1 complex. J. Immunol. 165:2048.-   Luo, C., N. M. Thielens, J. Gagnon, P. Gál, M. Sárvári, Y. Tseng, M.    Tosi, P. Závodszky, G. J. Arlaud, and V. N. Schumaker. 1992.    Recombinant human complement subcomponent C1s lacking    β-hydroxyasparagine, sialic acid, and one of its two carbohydrate    chains still reassembles with C1q and C1r to form a functional C1    complex. Biochemistry 31:4254.-   Matsushita, M. and Fujita, T. 1995. Cleavage of the third component    of complement (C3) by mannose-binding protein-associated serine    protease (MASP) with subsequent complement activation. Immunobiology    194:443.-   Matsushita, M. and T. Fujita. 1992. Activation of the classical    complement pathway by mannose-binding protein in association with a    novel C1s-like serine protease. J. Exp. Med. 176:1497.-   Matsushita, M., S. Thiel, J. C. Jensenius, I. Terai, and T.    Fujita. 2000. Proteolytic activities of two types of mannose-binding    lectin-associated serine protease. J. Immunol. 165:2637.-   McRae, B. J., T. Y. Lin, and J. C. Powers. 1981. Mapping the    substrate binding site of human C1r and C1s with peptide    thioesters. J. Mol. Biol. 256:12362.-   Monkovic, D. D., W. J. VanDusen, C. J. Petroski, V. M. Garsky, M. K.    Sardana, P. Závodszky, A. M. Stem, and P. A. Friedman. 1992.    Invertebrate aspartyl/asparaginyl β-hydroxylase: potential    modification of endogenous epidermal growth factor-like modules.    Biochem. Biophys. Res. Commun. 189:233.-   Monsinjon T, Richard V, Fontaine M. 2001. Complement and its    implications in cardiac ischemia/reperfusion: strategies to inhibit    complement. Fundam Clin Pharmacol 15(5):293-   Nagasawa, S, and R. M. Stroud (1977). Cleavage of C2 by C1s into the    antigenically distinct fragments C2a and C2b: demonstration of    binding of C2b to C4b. Proc Natl Acrid Sci USA 74: 2998-3001.-   Orsini, G., and M. L. Goldberg. 1978. The renaturation of reduced    chymotrypsinogen A in guanidin HCl. Refolding versus aggregation. J.    Biol. Chem. 253:3453.-   Pangburn, M. K. and H. I. Muller-Eberhard (1983). Kinetic and    thermodynamic analysis of the control of C3b by the complement    regulatory proteins factors H and I. Biochemistry 22: 178-85.-   pET System Manual TB055 7th Ed. 4/97. Novagen Inc., Madison-   Peterson, F. C., N. C. Gordon, and P. G. W. Gettins. 2001.    High-level bacterial expression and ¹⁵N-alanine-labeling of bovine    trypsin. Application to the study of trypsin-inhibitor complexes and    trypsinogen activation by NMR spectroscopy. Biochem. 40:6275.-   Pilatte, Y., C. H. Hammer, M. M. Frank and L. F. Fries (1989). A new    simplified procedure for C1-inhibitor purification. A novel use for    jacalin-agarose. J Immunol Methods 120: 37-43.-   Privalov, P. L. 1979. Stability of proteins: small globular    proteins. Adv. Protein Chem. 33:167.-   Protein Data Bank id code: 1elv-   Rossi, V., I. Bally, N. M. Thielens, A. F. Esser, and G. J.    Arlaud. 1998. Baculovirus-mediated expression of truncated modular    fragments from the catalytic region of human complement serine    protease C1s. J. Biol. Chem. 273:1232.-   Rossi, V., S. Cseh, I. Bally, N. M. Thielens, J. C. Jensenius,    and G. J. Arlaud. 2001. Substrate specificities of recombinant    mannan-binding lectin-associated serine proteases-1 and -2. J. Biol.    Chem. 276:40880.-   Rudolph, R and Lilie H (1996) In vitro folding of inclustion body    proteins. The Faseb Journal 10: 49-56-   Salama et al., N. Engl. J. Med. 318:408-14 (1988)-   Salvesen, G. and J. J. Enghild (1993). alpha-Macroglobulins:    detection and characterization. Methods Enzymol 223: 121-41.-   Sato T., Endo Y., Matsushita M., Fujita T.; 1994 Molecular    characterization of a novel serine protease involved in activation    of the complement system by mannose-binding protein. Int. Immunol.    6:665-669.-   Schumaker, V. N., P. Závodszky, and P. H. Poon. 1987. Activation of    the first component of complement. Annu. Rev. Immunol. 5:21.-   Sim, R. B. and A. Reboul (1981). Preparation and properties of human    C1 inhibitor. Methods Enzymol 80: 43-54.-   Sim, R. B., A. J. Day, B. E. Moffatt and M. Fontaine (1993).    Complement factor I and cofactors in control of complement system    convertase enzymes. Methods Enzymol 223: 13-35.-   Sim, R. B., and A. Laich. (2000). Serine proteases of the complement    system. Biochem. Soc. Trans. 28:545.-   Sim, R. B., Arlaud, G. J., and Colomb, M. G. (1980) Kinetics of    reaction of human C1-inhibitor with the human complement system    proteases C1r and C1s. Biochim. Biophys. Acta, 612, 433-449-   Soames, C. J. and R. B. Sim (1997). Interactions between human    complement components factor H, factor I and C3b, Biochem J326:    553-61.-   Stover, C. M., S. Thiel, M. Thelen, N. J. Lynch, T.    Vamp-Jensen, J. C. Jensenius, and W. Schwaeble. (1999). Two    constituents of the initiation complex of the mannan-binding lectin    activation pathway of complement are encoded by a single structural    gene. J. Immunol. 162:3481.-   Szilágyi, L., E. Kénesi, G. Katona, Gy. Kaslik, G. Juhasz, and L.    Gráf. 2001. Comparative in vitro studies on native and recombinant    human cationic trypsins. J. Biol. Chem. 276:24574.-   Tack, B. D. and J. W. Prahl (1976). Third component of human    complement: purification from plasma and physicochemical    characterization. Biochemistry 15: 4513-21.-   Takada F., Takayama Y., Hatsuse H., Kawakami M.; (1993). A new    member of the C1s family of complement proteins found in a    bactericidal factor, Ra-reactive factor, in human serum; Biochem.    Biophys. Res. Commun. 196:1003-1009-   Takahashi, M., Y. Endo, T. Fujita, and M. Matsushita. (1999). A    truncated form of mannose-binding lectin-associated serine protease    MASP-2 expressed by alternative polyadenylation is a component of    the lectin complement pathway. Int. Immunol. 11:859.-   Terai, I., K. Kobayashi, M. Matsushita, and T. Fujita. (1997), Human    serum mannose-binding lectin (MBL)-associated serine protease-1    (MASP-1): determination of levels in body fluids and identification    of two forms in serum. Clin. Exp. Immunol. 110:317.-   Thiel S., Jensen T. V., Stover C. M., Schwaeble W. J., Laursen S.    B., Poulsen K., Willis A. C., Eggleton P., Hansen S., Holmskov U.,    Reid K. B. M., Jensenius J. C. (1997) A second serine protease    associated with mannan-binding lectin that activates complement.    Nature 386:506-510.-   Thiel, S., S. V. Petersen, T. Vorup-Jensen, M. Matsushita, T.    Fujita, C. M. Stover, W. J. Schwaeble, and J. C. Jensenius. 2000.    Interaction of C1q and mannan-binding lectin (MBL) with C1r, C1s,    MBL-associated serine protease 1 and 2, and the MBL-associated    protein Map19. J. Immunol. 165:878.-   Thiel, S., T. Vorup-Jensen, C. M. Stover, W. Schwaeble, S. B.    Laursen, K. Poulsen, A. C. Willis, P. Eggleton, S. Hansen, U.    Holmskov, K. B. M. Reid, and J. C. Jensenius. 1997. A second serine    protease associated with mannan-binding lectin that activates    complement. Nature 386:506.-   Thijs et al., J. Immunol. 144:2419 (1990)-   Thielens, M. N., S. Cseh, S. Thiel, T. Vonap-Jensen, V. Rossi, J. C.    Jensenius, and G. J. Arlaud. 2001. Interaction properties of human    mannan-binding lectin (MBL)-associated serine protease-1 and -2,    MBL-associated protein 19, and MBL. J. Immunol. 166:5068.-   Thielens, N. M., M. Lacroix, J. Dobó, P. Gál, P. Závodszky,    and G. J. Arlaud. 1998. Expression and characterization of the    proenzyme and activated forms of the catalytic region of human C1r.    Mol. Immunol. 35:332.-   Turner, M. W. 1996. Mannose-binding lectin: the pluripotent molecule    of the innate immune system. Immunol. Today 17.532.-   Villiers, C. L., G. J. Arlaud, and M. G. Colomb. 1985. Domain    structure and associated functions of subcomponents C 1r and C I s    of the first component of human complement. Proc. Natl. Acad. Sci.    U.S.A. 82:4477.-   Volanakis, J. E., and G. J. Arlaud. 1998. Complement enzymes. In The    human Complement System in Health and Disease, M. Frank, and J. E.    Volanakis, eds. Marcel-Dekker, New York, p. 49.-   Vorup-Jensen, T., S. V. Petersen, A. G. Hansen, K. Poulsen, W.    Schwaeble, R. B. Sim, K. B. M. Reid, S. J. Davis, S. Thiel,    and J. C. Jensenius. 2000. Distinct pathways of mannan-binding    lectin (MBL)- and C1-complex autoactivation revealed by    reconstitution of MBL with recombinant MBL-associated serine    protease-2. J. Immunol. 165.2093.-   Wong, N. K. H., M. Kojima, J. Dobó, G. Ambrus, and R. B. Sim. 1999.    Activities of the MBL-associated serine proteases (MASPs) and their    regulation by natural inhibitors. Mol. Immunol. 36:853.-   Zhang, Y., C. Suankratay, D. R. Jones, X. H. Zhang, T. F. Lint,    and H. Gewurz. 1998. Lectin pathway hemolysis in the serum of the    guinea pig and other species. Mol. Immunol. 35:390.

1. Recombinant method for the preparation of an unglycosylated foldedC-terminal fragment of a multidomain serine protease, comprising thefollowing steps: a bacterial expression vector for expressing a DNAinsert encoding a C-terminal fragment of a multidomain serine proteaseis provided, said C-terminal fragment is produced in a bacterial host byusing the said vector and obtained in a folded form from the bacterialhost.
 2. The method of claim 1, wherein the multidomain serine proteaseencoded by the cDNA insert is a multidomain serine protease of thecomplement cascade, preferably having the following domain structureCUB-EGF-CUB-CCP1-CCP2-SP and being capable of binding to a recognitionmolecule of the complement pathway, e.g. to MBL or C1q, wherein saidserine protease is of vertebrata, preferably mammalian, more preferablyhuman origin.
 3. The method of claim 2, wherein the multidomain serineprotease C-terminal fragment encoded by the cDNA insert is a fragment ofcomprising one or more domain selected from the following domain types:CCP1, CCP2, SP, preferably, the C-terminal fragment has essentially adomain structure of any of the following: CCP1-CCP2-SP, CCP2-SP, SP. 4.The method of claim 3, wherein the C-terminal fragment is selected fromthe following fragments: MASP-1 CCP1-CCP2-SP fragment, MASP-2CCP1-CCP2-SP fragment, MASP-2 CCP2-SP fragment or MASP-2 SP fragment,MASP-3 CCP1-CCP2-SP fragment, MASP-3 CCP2-SP fragment or MASP-3 SPfragment, C1r CCP1-CCP2-SP fragment (i.e. γB-fragment), C1r CCP2-SPfragment or C1r SP fragment, C1s CCP1-CCP2-SP fragment, C1s CCP2-SPfragment or C1s SP fragment.
 5. The method of claim 4, wherein theC-terminal fragment comprises on its N-terminal a tag sequence thecoding sequence of which increases the efficiency of bacterial proteinexpression, preferably a sequence suitable for the promoter used forexpression.
 6. The method of claim 4, wherein said fragment comprisesmutation.
 7. The method of claim 4, wherein the bacterial host is E.coli and the expression vector is a vector capable of expressing,preferably overexpressing foreign genes in E. coli.
 8. The method ofclaim 1, wherein the step of obtaining the C-terminal fragment in afolded form comprises the following steps: i) the inclusion bodiesformed in the bacterial host are isolated ii) the protein fragmentmolecules are renatured from the inclusion bodies, iii) optionally, therenatured protein fragment molecules are further purified.
 9. The methodof claim 8, wherein the protein fragment is MASP-fragment, preferably aMASP-fragment selected from the following: MASP-1 CCP1-CCP2-SP fragment,MASP-2 CCP1-CCP2-SP fragment, MASP-2 CCP2-SP fragment or MASP-2 SPfragment, MASP-3 CCP1-CCP2-SP fragment, MASP-3 CCP2-SP fragment orMASP-3 SP fragment, and in the preparation method step c) abovecomprises the following steps: i) solubilization of the inclusionbodies, ii) diluting the solubilized protein fragments and transferringthem into a refolding buffer, iii) allowing the protein fragments torefold at a pH higher than 7, preferably above pH 8.5, more preferablybetween 8.5 and 10.5 and a temperature between 0 and 15° C., in anenvironment suitable for mildly oxidizing cysteine residues intocystines, iv) transferring the refolded protein fragments into anappropriate buffer.
 10. The method of claim 9, wherein in step i)solubilization is carried out in an appropriate solubilization agent,e.g. GuHCl or urea, preferably in a buffer comprising 6M GuHCl, anysuitable reducing agent e.g. β-mercaptoethanol or DTT, e.g. 100 mM DTT,and optionally a buffering agent, e.g. 0.1 M Tris-HCl (pH 8.3), e.g. atroom temperature or somewhat lower, the protein concentration in thesolution is preferably 0.5 to 25 mg/ml, more preferably 1 to 20 or 2 to10 mg/ml, even more preferably 3 to 7, e.g. about 5 mg/ml, and in stepii) the solubilized proteins are preferably diluted directly into therefolding buffer and the dilution is at least 10 fold, preferably atleast 100 fold, more preferably 200-600 fold, even more preferably about400 fold, prior dilution the refolding buffer is cooled below 10° C.,preferably below 5° C., more preferably to about 0° C. and in step iii),during refolding, the refolding buffer comprises a redox system, e.g.cysteine/cystine system or a glutathione system wherein the ratio of thereduced and oxidized forms is set to provide an mildly reductiveenvironment, the refolding buffer further comprises a chaotropic agentin an appropriate concentration, e.g. arginine (0.5 to 1.0 M). In caseof MASP-2, GuHCl (0.5 to 2.0 M), refolding is carried out at atemperature between 0 to 15° C., preferably between 0 to 10° C., morepreferably between 4 to 8° C.
 11. The method of claim 10, wherein theMASP protein fragment is MASP-1 CCP1-CCP2-SP fragment, MASP-2CCP1-CCP2-SP fragment or MASP-2 CCP2-SP fragment and the pH of therefolding buffer is between pH 8.5 and 9.5, preferably about pH 9.0. 12.The method of claim 10, wherein the MASP protein fragment is MASP-2 SPfragment and the pH is between pH 9.5 to 10.5, preferably pH
 10. 13. Themethod of claim 8, wherein the protein fragment is C1 serine proteasefragment, e.g. a C1s or a C1r fragment, and step c) of the preparationmethod comprises the following steps: i) solubilization of the inclusionbodies, ii) diluting the solubilized protein fragments and transferringthem into a refolding buffer, iii) allowing the protein fragments torefold at a pH higher than 5, preferably between 7 and pH 8.5 and atemperature between 0 and 15° C., in an environment suitable for mildlyoxidizing cysteine residues into cystines iv) transferring the refoldedprotein fragments into an appropriate buffer.
 14. The method of claim13, wherein in step i) solubilization is carried out in an appropriatesolubilization agent, e.g. GuHCl or urea, preferably in a buffercomprising 6M GuHCl, any suitable reducing agent e.g. β-mercaptoethanolor DTT, e.g. 100 mM DTT, and optionally a buffering agent, e.g. 0.1 MTris-HCl (pH 8.3), e.g. at room temperature or somewhat lower, theprotein concentration in the solution is preferably 0.5 to 25 mg/ml,more preferably 1 to 20 or 2 to 10 mg/ml, even more preferably 3 to 7,e.g. about 5 mg/ml, and In step ii) the solubilized proteins are diluteddirectly into the refolding buffer and the dilution is at least 10 fold,preferably at least 100 fold, more preferably 200-600 fold, even morepreferably about 400 fold, and in step iii), during refolding, therefolding buffer comprises a redox system, e.g. cysteine/cystine systemor a glutathione system, the refolding buffer further comprises achaotropic agent in an appropriate concentration, preferably GuHCl(preferably 2.0 M), refolding is carried out at a temperature between 0to 15° C., preferably between 4 to 15° C.
 15. A prokaryotic expressionvector, said vector comprising a DNA insert encoding a C-terminalfragment of a multidomain serine protease and means for expressing saidfragment in a bacterial host, wherein said serine protease is ofvertebrata, preferably mammalian, more preferably human origin.
 16. Theexpression vector of claim 15 which is a bacterial vector and the saidDNA insert is a cDNA insert, preferably encoding a fragment comprisingone or more domain selected from the following domain types: CCP1, CCP2,SP.
 17. The expression vector of claim 16, wherein the said cDNA insertencodes a fragment of a multidomain serine protease of the complementcascade (multidomain complement serine protease), preferably amultidomain serine protease selected from the following: MASP-1, MASP-2,MASP-3, C1r and C1q.
 18. The expression vector of claim 17, wherein thec-DNA insert encodes a C-terminal protein fragment having essentially adomain structure of any of the following: CCP1-CCP2-SP, CCP2-SP, SP. 19.The expression vector of claim 18, wherein the C-terminal fragment isselected from any of the following: MASP-1 CCP1-CCP2-SP fragment, MASP-2CCP1-CCP2-SP fragment, MASP-2 CCP2-SP fragment or MASP-2 SP fragment,MASP-3 CCP1-CCP2-SP fragment, MASP-3 CCP2-SP fragment or MASP-3 SPfragment, C1r CCP1-CCP2-SP fragment (i.e. γB-fragment), C1r CCP2-SPfragment or C1r SP fragment, C1s CCP1-CCP2-SP fragment (i.e.γB-fragment), C1s CCP2-SP fragment or C1s SP fragment.
 20. Theexpression vector of claim 19 wherein said fragment comprises amutation.
 21. The expression vector of claim 16 wherein the cDNA insertcomprises a sequence capable of increasing the efficiency of bacterialprotein expression, said sequence being operably linked to the sequenceencoding the protein fragment.
 22. A vector of any of the claims 15 to21, wherein the means for expressing the DNA insert sequence encodingany of the protein fragments as disclosed above, operably linked to apromoter, preferably an inducible overexpressing promoter capable ofdriving expression in a suitable bacterial host, preferably, thebacterial host is E. coli and the promoter is a promoter drivingoverexpression.
 23. An isolated C-terminal fragment of a multidomainserine protease of the complement cascade, obtained by the method of anyof claims 1 to 14, wherein said fragment is unglycosylated and folded.24. The isolated fragment of claim 23 wherein the multidomain serineprotease is a serine protease capable of binding to a recognitionmolecule of the complement pathway, e.g. MBL or C1q, said serineprotease having preferably the following domain structureCUB-EGF-CUB-CCP1-CCP2-SP, wherein said serine protease is of vertebrata,preferably mammalian, more preferably human origin.
 25. The isolatedfragment of claim 24 wherein the multidomain serine protease is selectedfrom the following: MASP-1, MASP-2, MASP-3, C1r, C1s.
 26. The isolatedfragment of claim 25, wherein the C-terminal fragment is selected fromany of the following: MASP-1 CCP1-CCP2-SP fragment, MASP-2 CCP1-CCP2-SPfragment, MASP-2 CCP2-SP fragment or MASP-2 SP fragment, MASP-3CCP1-CCP2-SP fragment, MASP-3 CCP2-SP fragment or MASP-3 SP fragment,C1r CCP1-CCP2-SP fragment (i.e. γB-fragment), C1r CCP2-SP fragment orC1r SP fragment, C1s CCP1-CCP2-SP fragment (i.e. γB-fragment), C1sCCP2-SP fragment or C1s SP fragment.
 27. The isolated fragment of claim26, wherein said fragment of the invention comprises on its N-terminal atag sequence the coding sequence of which increases the efficiency ofbacterial protein expression.
 28. The isolated fragment of claim 26which is autoactivated or capable of being autoactivated.
 29. Theisolated fragment of claim 26 wherein said fragment comprises mutation.30. The isolated fragment of claim 29 comprising a mutation selected oneor more from the following the activation site of the SP-domain ismutated, e.g. the Arg-Ile bond of the activation site is mutated,preferably to Gln-Ile, the active Ser is mutated, e.g. to Gly, Ala, Thr,Val, etc., preferably to Ala.
 31. Use of any of the C-terminalmultidomain complement serine protease fragments defined in any ofclaims 1 to 30, for raising antibodies.
 32. Use of any of the C-terminalmultidomain complement serine protease fragments defined in any ofclaims 1 to 30, as a standard or a control in assessing the level oractivity of a multidomain complement serine protease in a biologicalsample.
 33. Use of the C-terminal multidomain complement serine proteasefragments defined in any of claims 1 to 30, for screening substrates orinhibitors of MASP-1 or MASP-2 or MASP-3, C1r or C1s.
 34. An assaymethod for measuring the level of a multidomain complement serineprotease in a biological sample, wherein the presence of said serineprotease is quantitatively detected in the sample by a labeledmonoclonal antibody and the obtained signal is compared with a signalobtained for a control sample comprising a respective complementprotease fragment according the invention.
 35. An assay method formeasuring the activity of a multidomain complement serine protease in abiological sample, wherein the activity on a substrate of said proteaseis measured, and an appropriate fragment, preferably a CCP1-CCP2-SPfragment, of said protease according to the invention is used as astandard with the proviso that it has the same specific activity as therespective protease or the ratio of the activities of the nativeprotease and the fragment is known.
 36. An assay method for assessingMASP-1 and MASP-2 levels in a sample of biological origin, said methodcomprising monitoring C2 cleavage and C4 cleavage by MASP proteins inaliquots of the sample whereas, if desired, other complement pathwaysare blocked, considering C4 conversion as a result of MASP 2 activityand C2 conversion as a result of MASP-1 and MASP-2 activity togethercalculating MASP-1 and MASP-2 levels using either known specificactivity values of said proteins or MASP-1 and MASP-2 CCP1-CCP2-SPfragments, respectively, as inner standards.
 37. An assay method forassessing MASP-1 and MASP-2 levels in a sample of biological origin,said method comprising monitoring C2 cleavage in the sample andconsidering C2 conversion as a result of MASP-1 and MASP-2 activitytogether, adding a calculated amount of α2M to the sample to inhibitMASP-1 activity but leaving MASP-2 activity unchanged or changing it toa negligible or a calculable extent, monitoring C2 activity in thesample comprising α2M, calculating MASP-1 and MASP-2 levels using eitherknown specific activity values of said proteins or MASP-1 and MASP-2CCP1-CCP2-SP fragments, respectively, as inner standards.
 38. Adiagnostic kit for carrying out an assay method of any of claims 34 to37, said kit comprising a fragment defined in any of claims 1 to
 30. 39.Use of a MASP-1 fragment defined in any of claims 1 to 30 for inducingblood coagulation.
 40. Use of an inhibitor of MASP-1, preferably α2M orC1-inhibitor, for reducing blood coagulation.
 41. Method for treatmentof a patient in need of inhibiting complement activity exerted throughthe lectin pathway, preferably reperfusion injury, comprisingadministering any inhibitor of the lectin complement pathway, preferablyan inhibitor of MASP-2, to the patient in an effective quantity.